Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus.

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Banana Peels: A Promising Substrate for the Coproduction of Pectinase and Xylanase from Aspergillus fumigatus MS16.

Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30-35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30­35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate.

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 106 spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.

Biosynthesis, purification and characterization of ß-1,4-xylanase from a novel mangrove associated actinobacterium Streptomyces olivaceus (MSU3) and its applications.

The present study was undertaken to evaluate the xylanolytic properties of an actinobacterium Streptomyces olivaceus (MSU3) isolated from the sediment sample of mangrove environment. It showed highest xylanase activity on initial screening in Breg's mineral salts medium supplemented with 0.5% xylan. Further the organism expressed maximum xylanase production at optimized culture conditions of pH 7, temperature 30 °C with 2.5% of inoculum size at 72 h of incubation, the nutrient sources like sucrose (2%) and yeast extract (3%) have observed as best carbon and nitrogen sources respectively. In purification, the xylanase resulted 4.27fold increase with the yield of 15.57% at the final step using sephadex G-75 chromatography. The molecular weight of the purified xylanase was observed as 42 kDa on 10% SDS-PAGE and further identified by MALDI-TOF-MS analysis. It belongs to GH43 family encoded 485 amino acid residues and the xylanase gene isolated using PCR amplification was partially sequenced. It showed 98% sequence similarity to the xylanase gene of S. olivaceus. The maximum activity of purified xylanase was observed at pH 8, temperature 40 °C and also the production medium substituted with Fe3+ metal ion, 2.0% xylan and 1.5% NaCl along with Km and Vmax values of 8.16 mg/ml and 250.01 µg/min/mg, respectively. In xylanolytic hydrolysis of pretreated agro-wastes, especially the sugarcane juice substituted medium yielded maximum (52.19%) reducing sugar, followed by bioethanol production (4.19  g/L) at 72 h of incubation. Based on the results, it could be confirmed that the selected isolate is a potent strain and it can able to produce xylanase through fermentation process and also it can able to convert the pretreated agro-wastes into economically important byproduct like bioethanol.

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK.

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315 ± 16 IU/mL acidic xylanase, 290 ± 20 IU/mL alkaline xylanase, and 88 ± 9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10 mM MgSO4, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60 hr at 200 rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3 hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6 hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.

Production of thermo-alkali-stable laccase and xylanase by co-culturing of Bacillus sp. and B. halodurans for biobleaching of kraft pulp and deinking of waste paper.

To reduce pollution and cost of treatment for fresh and recycled paper, co-production of xylanase and laccase was carried out in the same production medium using two compatible species of Bacillus. These co-produced enzymes were used for deinking of old newsprint (ONP) and biobleaching of eucalyptus Kraft pulp. Solid-state co-cultivation of Bacillus sp. and B. halodurans FNP135 was optimized statistically by response surface methodology for the co-production of xylanase (X) and laccase (L). A significant increase in production of xylanase (2.1-fold, 1,685 IU/g) and laccase (2.04-fold, 2,270 nkat/g) was observed under optimized conditions viz. pH (10.5), inoculum size (10 + 10 %) and moisture:substrate ratio (0.8:1). Both the enzymes showed identical temperature and pH optima of 70 °C and 9, respectively, and were used for deinking of ONP pulp and biobleaching of kraft pulp. In case of ONP pulp deinking, the XL treatment increased brightness (11.8 %), freeness (17.8 %), breaking length (34.8 %), burst factor (2.77 %) and tear factor (2.4 %). In case of kraft pulp biobleaching, XL treatment showed a significant increase in brightness (13 %), whiteness (106.15 %) breaking length (49 %), burst factor (6.9 %), tear factor (23 %), and viscosity (11.68 %) and reduction in kappa number (15 %) after alkali extraction and peroxide stage. This enhancement of pulp properties revealed a synergistic effect of xylanase and laccase produced in one setup.

Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis.

De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries.

Optimization of fibrolytic enzyme production by Aspergillus japonicus C03 with potential application in ruminant feed and their effects on tropical forages hydrolysis.

Fibrolytic enzyme production by Aspergillus japonicus C03 was optimized in a medium containing agro-industrial wastes, supplemented with peptone and yeast extract. A 2(3) full factorial composite and response surface methodology were used to design the experiments and analysis of results. Tropical forages were hydrolyzed by A. japonicus C03 enzymatic extract in different levels, and they were also tested as enzymatic substrate. Optimal production to xylanase was obtained with soybean bran added to crushed corncob (1:3), 0.01% peptone, and 0.2% yeast extract, initial pH 5.0, at 30 °C under static conditions for 5 days of incubation. Optimal endoglucanase production was obtained with wheat bran added to sugarcane bagasse (3:1), 0.01% peptone, and 0.2% yeast extract, initial pH 4.0, at 30 °C, for 6 days, under static conditions. Addition of nitrogen sources as ammonium salts either inhibited or did not influence xylanase production. This enzymatic extract had a good result on tropical forage hydrolyzes and showed better performance in the Brachiaria genera, due to their low cell wall lignin quantity. These results represent a step forward toward the use of low-cost agricultural residues for the production of valuable enzymes with potential application in animal feed, using fermentation conditions.

Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization.

Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains.

Two strains of Pleurotus ostreatus (IE-8 and CP-50) were grown on defined medium added with wheat straw extract (WSE). Mycelia from these cultures were used as an inoculum for solid fermentation using sugar cane bagasse (C:N=142). This substrate was used separately either as a mixture of heterogeneous particle sizes (average size 2.9 mm) or as batches with two different particle sizes (0.92 mm and 1.68 mm). Protein enrichment and production of lignocellulolytic enzymes on each particle size was compared. The effect of ammonium sulphate (AS) addition was also analyzed (modified C:N=20), this compound favored higher levels of protein content. Strain CP-50 showed the highest increase of protein content (48% on particle size of 1.68 mm) when compared to media with no additional N source. However, strain IE-8 produced the highest levels of all enzymes: xylanases (5.79 IU/g dry wt on heterogeneous particles) and cellulases (0.18 IU/g dry wt on smallest particles), both without the addition of AS. The highest laccase activity (0.040 IU/g dry wt) was obtained on particles of 1.68 mm in the presence of AS. Since effect of particle size and addition AS was different for each strain, these criteria should be considered for diverse biotechnological applications.

Phytase production by thermophilic mold Sporotrichum thermophile in solid-state fermentation and its application in dephytinization of sesame oil cake.

The phytase production by Sporotrichum thermophile TLR50 was recorded on all the commonly used animal feed ingredients tested to varying degrees in solid-state fermentation. Enzyme production increased to 180 U/g of dry moldy residue (DMR) in sesame oil cake at 120 h and 45 degrees C at the initial substrate-to-moisture ratio of 1:2.5 and aw of 0.95. Supplementation of sesame oil cake with glucose and ammonium sulfate further enhanced phytase titer (282 U/g of DMR). An overall 76% enhancement in phytase production was achieved owing to optimization. The mold secreted acid phosphatase, amylase, xylanase, and lipase along with phytase. By the action of phytase, inorganic phosphate was liberated efficiently, leading to dephytinization of sesame oil cake.

Profile of enzyme production by Trichoderma reesei grown on corn fiber fractions.

Corn fiber is the fibrous by-product of wet-mill corn processing. It typically consists of about 20% starch, 14% cellulose, and 30% hemicellulose in the form of arabinoxylan. Crude corn fiber (CCF) was fractionated into de-starched corn fiber (DSCF), corn fiber with cellulose (CFC) enriched, and corn fiber arabinoxylan (CFAX), and these fractions were evaluated as substrates for enzyme production by Trichoderma reesei. T. reesei QM9414 and Rut C-30 grew on CCF, DSCF, CFC, or CFAX and secreted a number of hydrolytic enzymes. The enzymes displayed synergism with commercial cellulases for corn fiber hydrolysis.

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium.

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.

Extractive cultivation of xylanase by Penicillium janthinellum in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system.

Cultivation of the fungus Penicillium janthinellum for xylanase production was studied in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system, using a two-level fractional factorial design. The parameters studied were initial pH, cultivation time, type of agro-industrial residue (oat husk or corn cob), agitation, temperature, and phase-forming polymers. The xylanase produced during fermentation partitioned into the top phase. The agitation and temperature (negative), cultivation time and initial pH (positive) effects proved statistically significant for xylanase production. The highest percentage yield of the xylanase in the top and its production in the top phase, about 97% and 160.7 U/mL, were obtained in cultures of 120 h, 40 rpm, 25 degrees C, and pH 5.0.