Newly Isolated Penicillium sp. for Cellulolytic Enzyme Production in Soybean Hull Residue

Abstract (1) Background: The aim of this study was to evaluate the production and partial characterization of xylanase and avicelase by a newly isolated Penicillium sp. in solid-state fermentation, using soybean hulls as substrate. (2) Methods: Temperature, time, number of spores, and substrate moisture on xylanase and avicelase bioproduction were evaluated, maximizing activity with 30°C, 1x106 spores/g substrate, 14 and 7 days of fermentation with 70 and 76% substrate moisture contents, for xylanase and avicelase, respectively. (3) Results: Different solvents, temperatures, and agitation in the enzymatic extraction were evaluated, obtaining higher activities, 430.77 and 26.77 U/g for xylanase and avicelase using 30 min extraction and 0.05 M citrate buffer solution (pH 4.5 ), respectively at 60°C and 175 rpm and 50°C and 125 rpm. The optimum pH and temperature for enzymatic activity determination were 5.3 and 50°C. Enzyme extract stability was evaluated, obtaining higher stability with pH between 4.5 and 5.5, higher temperature of up to 40°C. The kinetic thermal denaturation (Kd), half-life time, D-value, and Z-value were similar for both enzymes. The xylanase Ed value (89.1 kJ/mol) was slightly lower than the avicelase one (96.7 kJ/mol), indicating higher thermostability for avicelase. (4) Conclusion: In this way, the production of cellulases using alternative substrates is a way to reduce production costs, since they represent about 10% of the world demand of enzymes, with application in animal feed processing, food production and breweries, textile processing, detergent and laundry production, pulp manufacturing and the production of biofuels.

EcXyl43 ß-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

An ascomycota coculture in batch bioreactor is better than polycultures for cellulase production.

Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, ß-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, ß-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and ß-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.

The DNA-methyltransferase inhibitor 5-aza-2-deoxycytidine affects Humicola grisea enzyme activities and the glucose-mediated gene repression.

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification.

AIMS: To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification. METHODS AND RESULTS: Thirty-two heat-tolerant fungi were isolated from the environment, identified (morphological/molecular tools) and the production of the enzymes was evaluated by solid state fermentation using lignocellulosic materials as substrates. Myceliophthora thermophila JCP 1-4 was the best producer of endoglucanase (357·51 U g(-1) ), ß-glucosidase (45·42 U g(-1) ), xylanase (931·11 U g(-1) ) and avicelase (3·58 U g(-1) ). These enzymes were most active at 55-70°C and stable at 30-60°C. Using crude enzymatic extract from M. thermophila JCP 1-4 to saccharify sugarcane bagasse pretreated with microwaves and glycerol, glucose and xylose yields obtained were 15·6 and 35·13% (2·2 and 1·95 g l(-1) ), respectively. CONCLUSIONS: All isolated fungi have potential to produce the enzymes; M. thermophila JCP 1-4 enzymatic extract have potential to be better explored in saccharification experiments. Pretreatment improved enzymatic saccharification, as sugar yields were much higher than those obtained from in natura bagasse. SIGNIFICANCE AND IMPACT OF THE STUDY: Myceliophthora thermophila JCP 1-4 produces avicelase (not commonly found among fungi; important to hydrolyse crystalline cellulose) and a ß-glucosidase resistant to glucose inhibition, interesting characteristics for saccharification experiments.

Analysis of inducers of xylanase and cellulase activities production by Ganoderma applanatum LPB MR-56.

This manuscript describes the analysis of the effect of cellulose, carboxymethylcellulose (CMC), xylan, and xylose as inducers of cellulase and xylanase activity production by Ganoderma applanatum MR-56 and the optimization of their production in liquid cultures by statistical methods. The Plackett-Burman screening design was applied to identify the most significant inducers of xylanase and cellulase activities production by G. applanatum MR-56. The most significant effect on xylanase and cellulase activities production was exercised by cellulose, even if xylose and CMC were also effective at some times. The combined effect of cellulose, yeast extract, and pH was analyzed by a 2(3) factorial experimental design with four central points that showed that the maximum tested cellulose (1 % w/v) and yeast extract (5 g L(-1)) concentrations gave the maximum production of xylanase (8.24 U mL(-1)) and cellulase (3.29 U mL(-1)) activity at pH 6 and 4, respectively. These values achieved for cellulase and xylanase activity represent 12-25 fold and 36 fold higher values than the maximum so far reported for other strains of G. applanatum, respectively.

Penicillium purpurogenum produces two GH family 43 enzymes with ß-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.

ß-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with ß-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-ß-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, ß-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3-pNPXyl, ABF3-pNPAra and XYL1-pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1-pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.

D-xylose-fermenting and xylanase-producing yeast species from rotting wood of two Atlantic Rainforest habitats in Brazil.

This study investigated the yeast species associated with rotting wood in Brazilian Atlantic Rainforest ecosystems focusing on the identification of D-xylose-fermenting and/or xylanase-producing species. A total of 321 yeast strains were isolated from rotting wood samples collected in two Atlantic Rainforest areas. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Schwanniomyces polymorphus, Scheffersomyces queiroziae, Barnettozyma californica, and Candida (Ogataea) boidinii were the most frequently isolated yeasts. The rarefaction curves for the yeast communities isolated in YNB-D-xylose and YNB-xylan from both areas continued to rise and did not reach an asymptote, indicating that not all yeast diversity had been recovered. Additionally, the yeast composition was variable among the samples and areas, which was confirmed by the values of the Sorensen index. Among the 69 species identified, only 12 were found in both areas sampled. Fifteen possible new species were obtained. Among them, two species (Sugiyamaella sp. 1 and Sugiyamaella xylanicola) showed the ability to ferment D-xylose into ethanol, and three species (Spencermartinsiella sp. 1, Su. xylanicola and Tremella sp.) were able to produce extracellular xylanases. Indeed, most of the xylanase-producing isolates belong to the new species Su. xylanicola, which was also positive for D-xylose fermentation. S.queiroziae and S. stipitis were the main D-xylose-fermenting yeasts identified. The results of this work showed that rotting wood collected from the Atlantic Rainforests is a huge source of yeasts, including new species, with promising biotechnological properties.

Screening and xylanase production by Streptomyces sp. grown on lignocellulosic wastes.

Xylanases have raised interest because of their potential applications in various industrial fields, including the pulp and paper industries, bioethanol production, and the feed industry. In bioethanol production from lignocellulosic compounds, xylanase can improve the hydrolysis of cellulose into fermentable sugars, since the xylan restricts the cellulases from acting efficiently. In this work, a new thermophilic Streptomyces sp. was selected for its ability to produce xylanase. Carbon source selection is an important factor in the production of hemicellulases. The highest activity was obtained when Streptomyces sp. I3 was grown in the presence of wheat bran. Xylanase activity was partially characterized concerning the effect of pH and temperature on activity and thermostability, and the effects of different metal ions were also tested. The pH and temperature profile showed optimal activity at pH 6.0/70 °C. Zymogram analysis showed multiple xylanases (39, 21, 18, and 17 kDa). Xylanases studied in this work are thermophilic, thermostable, and active in a wide pH range; they have potential to be used in the development of new processes of biotechnological interest.

Expression and characterization of a GH39 ß-xylosidase II from Caulobacter crescentus.

In the present work, the gene xynB2, encoding a ß-xylosidase II of the Glycoside Hydrolase 39 (GH39) family, of Caulobacter crescentus was cloned and successfully overexpressed in Escherichia coli DH10B. The recombinant protein (CcXynB2) was purified using nickel-Sepharose affinity chromatography, with a recovery yield of 75.5 %. CcXynB2 appeared as a single band of 60 kDa on a sodium dodecyl sulfate polyacrylamide gel and was recognized by a specific polyclonal antiserum. The predicted CcXynB2 protein showed a high homology with GH39 ß-xylosidases of the genus Xanthomonas. CcXynB2 exhibited an optimal activity at 55 °C and a pH of 6. CcXynB2 displayed stability at pH values of 4.5-7.5 for 24 h and thermotolerance up to 50 °C. The K (M) and V (Max) values were 9.3 ± 0.45 mM and 402 ± 19 µmol min(-1) for ρ-nitrophenyl-ß-D-xylopyranoside, respectively. The purified recombinant enzyme efficiently produced reducing sugars from birchwood xylan and sugarcane bagasse fibers pre-treated with a purified xylanase. As few bacterial GH39 family ß-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.

Lichtheimia blakesleeana as a new potencial producer of phytase and xylanase.

Brazil is known for its great potential for production of renewable resources such as agro-industrial residues. These residues can be used as alternative sources of new products. Meanwhile, solid-state fermentation, with its advantages of energy conservation and pollution reduction, has been identified as a process of great potential for the production of bioactive compounds, especially enzymes. In the present work, a 2(3) factorial design was used to evaluate the effects of pH, temperature and moisture on the production of phytase and xylanase by Lichtheimia blakesleeana URM 5604 through the fermentation of citrus pulp. Statistical analyses of the results showed that the only the pH influenced the production of these enzymes, with the best phytase production (264.68 U/g) ocurring at pH 6.0, 34 °C, initial moisture 50%, after 48 hours of culture. The best conditions for xylanase production (397.82 U/g) were fermentation for 120 hours at pH 4.0, 26 °C and initial moisture of 70%. The best parameters for the simultaneous production of phytase (226.92 U/g) and xylanase (215.59 U/g) were determined to be initial moisture of 50%, pH 6.0, 26 °C, and 48 hours of fermentation.

Production of xylanolytic enzymes by Penicillium janczewskii.

The production of extracellular xylanase, beta-xylosidase and alpha-l-arabinofuranosidase by the mesophilic fungus Penicillium janczewskii under submerged cultivation was investigated with different carbon sources. Optimization steps included studies of carbon source concentration, temperature of cultivation and initial pH of culture medium. The production of these enzymes was increased two times when cultures were supplemented with brewer's spent grain at 2% concentration, pH 6.0 and carried out at 25 degrees C. Under these optimized conditions were obtained xylanase activity of 15.19UmL(-1) and 23.54Umgprot(-1), beta-xylosidase activity of 0.16UmL(-1) and 0.25Umgprot(-1) and alpha-l-arabinofuranosidase activity of 0.67UmL(-1) and 1.04Umgprot(-1). Brewer's spent grain is a promising substrate for P. janczewskii growth and xylanolytic enzyme production, since it is the main by-product from the brewing industry, available in large amounts and at low-cost in many countries.

Cell-associated acid beta-xylosidase production by Penicillium sclerotiorum.

In recent decades, beta-xylosidases have been used in many processing industries. In this work, the study of xylosidase production by Penicillium sclerotiorum and its characterization are reported. Optimal production was obtained in medium supplemented with oat spelts xylan, pH 5.0, at 30 degrees C, under stationary condition for six days. The optimum activity temperature was 60 degrees C and unusual optimum pH 2.5. The enzyme was stable at 50 and 55 degrees C, with half-life of 240 and 232min, respectively. High pH stability was verified from pH 2.0 to 4.0 and 7.5. The beta-xylosidase was strongly inhibited by divalent cations, sensitive to denaturing agents SDS, EDTA and activated by thiol-containing reducing agents. The apparent V(max) and K(m) values was 0.48micromol PNXPmin(-1)mg(-1) protein and 0.75mM, respectively. The enzyme was xylose tolerant with a K(i) of 28.7. This enzyme presented interesting characteristics for biotechnological process such as animal feed, juice and wine industries.

Beta-methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena.

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. beta-Methyl-xyloside (beta-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with beta-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus beta-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus beta-mx as substrate. To our knowledge, this is the first report on the effect of beta-mx on the induction of xylanase in C. flavigena.

Growth and pectinase production by Aspergillus Mexican strain protoplast regenerated under acidic stress.

Protoplasts from Aspergillus sp. FP-180 and Aspergillus awamori NRRL- 3112 were released and regenerated at extreme acidic conditions. The best conditions for protoplast release were 0.8 M KCI, pH 5.8, and 3 h of digestion using mycelia from 12- to 16-h cultures from either Aspergillus sp. FP-180 or A. awamori NRRL-3112. The addition of fresh mycelia to an ongoing digestion after 1 h increased protoplast 4.5-5 times. A regeneration efficiency of 90% was attained at pH 6.0, and it was possible to regenerate protoplasts at pH 1.7 with a regeneration efficiency of 0.5% for Aspergillus sp. FP-180. The LpH-10 strain, derived from protoplast from Aspergillus sp FP-180, was able to regenerate at pH 1.7 and grow at pH values as low as 1.5, values at which the original strain is unable to grow. Regeneration at extreme pH improved the performance of LpH-10 strain. It showed a twofold increase in cell growth at pH 2.0 in liquid culture and a higher pectinolytic activity in relation to that produced by the original strain.

Kinetics of the solid state fermentation of sugarcane bagasse by Thermoascus aurantiacus for the production of xylanase.

Xylanase was produced by solid-state fermentation using Thermoascus aurantiacus. Maximum production (500 U g(-1) bagasse) was achieved on the sixth day of cultivation on solid sugarcane bagasse medium supplemented with 15% (v/w) rice bran extract. The fungal biomass, determined from its glucosamine content, reached 28 mg g(-1) on the 8th day of cultivation. The cell yield against O2 (Y(x/o) = 0.18 g(cell)/g(O2)) and maintenance coefficient (m0 = 0.013 g(O2)/g(cell)h) were determined with the low Y(x/o) value for T. aurantiacus agreeing with the calculated value.

Xylanolytic complex from Aspergillus giganteus: production and characterization.

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 degrees C and under shaking at 120 rpm during 84 h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 degrees C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T(50) of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 degrees C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg(+2), Cu(+2) and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and beta-xylosidase activity in assay conditions.

Xylanase production by Trichoderma reesei rut C-30 on rice straw.

Xylanase production of Trichoderma reesei Rut C-30 was examined at different initial pH values (4.8, 5.9, and 7.0) on rice straw in shake flasks, and in a fermentor, for the best pH condition. Enzyme performance was tested on ammonia-treated dwarf elephant grass. The maximum xylanase activities, 92 and 122 IU/mL, were obtained at pH 4.8 in the shake flasks and fermentor, respectively, in which good growth of the fungus was observed during the first 24 h and consumption of proteins dissolved from the rice straw caused the pH to rise later to values between 6.4 and 6.7 (optimal for xylanase production). The xylanases from T. reesei were as effective as Multifect XL, a commercial enzyme preparation, in hydrolyzing ammonia-treated elephant grass.

Temperature effect in the production of multiple xylanases by Aspergillus fumigatus.

This work has evaluated the temperature effect in the production of multiple xylanases by a locally isolated strain of Aspergillus fumigatus Fresenius. Three isoenzymes, identified as xylanases I, II, and III with apparent molecular weight of 45.7 KDa, 39.8 KDa and 18.2 KDa, respectively, were produced in cultures developed at 30 degrees C and at 42 degrees C. The pattern of distribution of xylanase activity among the three isoenzymes was greatly affected by the growth temperature: at 30 degrees C, the total xylanase activity was distributed homogeneously among the three enzymes, while at 42 degrees C, the total xylanase activity was mainly due to the fractions with the highest MW (I and II) and the xylanase III was a minor component.

Draw-fill batch culture mode for production of xylanases by Cellulomonas flavigena on sugar cane bagasse.

Draw-fill culture was evaluated as a method for xylanase production by Cellulomonas flavigena on sugar cane bagasse. Specific xylanase activity and volumetric xylanase activities were measured by harvesting 50%, 55%, 60% and 70% of fermented broth at the end of each subculture. Maximum specific (64 IU mg(-1) protein) and volumetric (166 IU ml(-1)) xylanase activities were obtained by harvesting 50-55% of broth. Values were 3.4 and 3.8 times greater than those obtained in batch cultures carried out under the same conditions. Enzyme productivity of 4.2 IU ml(-1) h(-1) was significantly greater than that obtained in continuous cultures (2.4 IU ml(-1) h(-1)) (P<0.05).