Screening and Production Study of Microbial Xylanase Producers from Brazilian Cerrado

Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by β-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 °C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0–5.5. They were stable in the pH range 5.0–10.0 and 5.5–8.5 for bacterial and fungal xylanase, respectively. The optimum temperatures were 55C and 60 °C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 °C.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Cold-Active Xylanase Produced by Fungi Associated with Antarctic Marine Sponges

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5–7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.     

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00?±?0.15 and 0.21?±?0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH?7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.     

A Novel Low Molecular Weight Endo-xylanase from Streptomyces sp. CS628 Cultivated in Wheat Bran

An extracellular low molecular weight xylanase (Xyn628) from Streptomyces sp. CS628 was isolated from Korean soil sample, produced in wheat bran medium, purified, and biochemically characterized. Xyn628 was purified 4.8-fold with a 33.78 % yield using Sepharose CL-6B column chromatography. The purified xylanase was ~18.1 kDa estimated by SDS-PAGE and xylan zymography. N-terminal amino acid sequences of Xyn628 were AYIKEVVSRAYM. The enzyme was found to be stable in a broad range of pH (5.0–13.0) and up to 60 °C and have optimal pH and temperature of pH 11.0 and 60 °C, respectively. Xyn628 activities were remarkable affected by various detergents, chelators, modulators, and metal ions. The xylanase produced xylobiose and xylotriose as principal hydrolyzed end products from the xylan. It was found to degrade agro-waste materials like corn cob and wheat bran by Xyn628 (20 U/g) as shown by electron microscopy. As being simple in purification, low molecular weight, alkaline, thermostable, and ability to produce xylooligosaccharides show that Xyn628 has potential applications in bioindustries as a biobleaching agent or/and xylooligosaccharides production with an appropriate utilization of agro-waste.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

The Effects of Wheat Bran Composition on the Production of Biomass-Hydrolyzing Enzymes by Penicillium decumbens

The effects of the starch, protein, and soluble oligosaccharides contents in wheat bran on the extracellular biomass-hydrolyzing enzymes activities released by Penicillium decumbens mycelia grown in batch fermentations have been examined. The results showed increased starch content correlated directly with an increase in released amylase activity but inversely with the levels of secreted cellulase and xylanase. High amounts of protein in wheat bran also reduced the activities of cellulase, xylanase and protease in the culture medium. The effects of the soluble and insoluble components of wheat bran and cello-oligosaccharides supplements on production of extracellular cellulase and xylanase were compared. The soluble cello-oligosaccharides compositions in wheat bran were proved to be one of the most significant factors for cellulase production. According to the results of this research, determining and regulating the composition of wheat bran used as a fermentation supplement may allow for improved induction of cellulase and xylanase production.     

Phytase Production by Aspergillus oryzae in Solid-State Fermentation and its Applicability in Dephytinization of Wheat Ban

Aspergillus oryzae SBS50 secreted a high titre of phytase in solid-state fermentation (SSF) using wheat bran at 30 °C after 96 h at the initial substrate to moisture ratio of 1:2 and a water activity of 0.95. The production of phytase increased when wheat bran was supplemented with sucrose and beef extract. Further enhancement in enzyme production was recorded when the substrate was supplemented with the surfactant Triton X-100 (145 U/g of DMB). An overall 29-fold improvement in phytase production was achieved owing to optimization. Under optimized conditions, the mould secreted 9.3-fold higher phytase in SSF as compared to submerged fermentation (SmF). The mesophilic mould also secreted amylase, cellulase (CMCase), pectinase and xylanase along with phytase in SSF. Scanning electron microscopy revealed luxuriant growth of A. oryzae on wheat bran with abundant spores. The enzyme dephytinized wheat bran with concomitant liberation of inorganic phosphate.     

Production of Crude Cellulase and Xylanase From Trichoderma harzianum PPDDN10 NFCCI-2925 and Its Application in Photocopier Waste Paper Recycling

This paper implies production of cellulase and xylanase enzyme using a potent strain of Trichoderma harzianum for the efficient deinking of photocopier waste papers. Different nutritional and environmental factors were optimized for higher production of cellulase along with xylanase. After fermentation, maximum enzyme extraction was achieved from fermented matter using a three-step extraction process with increased efficiency by 26.6–29.3 % over single-step extraction. Static solid state was found as the best fermentation type using wheat bran (WB) as carbon source and ammonium ferrous sulfate (0.02 M) as nitrogen source. Subsequently, inoculum size (8?×?10⁶ CFU/gds), incubation days (4 days), temperature (34 °C), initial pH (6.0), and moisture ratio (1:3) significantly affected the enzyme production. Cellulase and xylanase activities were found to be maximum at pH 5.5 and temperature 55–60 °C with good stability (even up to 6 h). Furthermore, this crude enzyme was evaluated for the deinking of photocopier waste papers without affecting the strength properties with improved drainage as an additional advantage. The crude enzyme-deinked pulp showed 23.6 % higher deinking efficiency and 3.2 % higher brightness than chemically deinked pulp. Strength properties like tensile, burst indices, and folding endurance were also observed to improve by 6.7, 13.4, and 10.3 %, respectively, for enzyme-deinked pulp. However, the tear index was decreased by 10.5 %. The freeness of the pulp was also increased by 21.6 % with reduced drainage time by 13.9 %.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Influences of Media Compositions on Characteristics of Isolated Bacteria Exhibiting Lignocellulolytic Activities from Various Environmental Sites

Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.     

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5–6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6–10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.     

Production of Cellulolytic Enzymes by Fungi Acrophialophora nainiana and Ceratocystis paradoxa Using Different Carbon Sources

Although a number of filamentous fungi, such as Trichoderma and Aspergillus, are well known as producers of cellulases, xylanases, and accessory cellulolytic enzymes, the search for new strains and new enzymes has become a priority with the increase in diversity of biomass sources. Moreover, according to the type of pretreatment applied, biomass of the same type may require different enzyme blends to be efficiently hydrolyzed. This study evaluated cellulases, xylanases, and β-glucosidases produced by two fungi, the thermotolerant Acrophialophora nainiana and Ceratocystis paradoxa. Cells were grown in submerged culture on three carbon sources: lactose, wheat bran, or steam-pretreated sugarcane bagasse, a commonly used cattle feed in Brazil. Xylanase and endo-1-4-β-glucanase (CMCase) highest production were found in A. nainiana growing on lactose and reached levels of 2,200 and 2,016 IU/L, respectively. C. paradoxa showed highest activity for xylanase when grown on wheat bran and for β-glucosidase when grown on steam-treated bagasse, at levels of 12,728 and 1,068 IU/mL, respectively.     

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.     

Lactose-inducted production of a complete lignocellulolytic enzyme system by a novel bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. BS-5 and its application for saccharification of alkali-pretreated corn cob

In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass.     

Evaluation of Enzyme Activity and Fiber Content of Soybean Cotyledon Fiber and Distiller’s Dried Grains with Solubles by Solid State Fermentation

To increase the value of coproducts from corn ethanol fermentation and soybean aqueous processing, distiller??s dried grains with solubles (DDGS) and soybean cotyledon fiber were used as the substrates for solid state fermentation (SSF) to improve feed digestibility. Aspergillus oryzae, Trichoderma reesei, and Phanerochaete chrysosporium were chosen as they produce desirable enzymes and are widely used in SSF for feed. The results showed that the cellulase and xylanase activities were significantly increased after 7?days of fermentation, and cellulose and hemicellulose degradation was also greatly increased. When soybean fiber was used as SSF substrate, the maximum activities of the cellulase and xylanase were 10.3 and 84.2?IU/g substrate (dry weight basis) after SSF treatment, respectively. However, the enzyme activities were relatively low in DDGS, and the growth of the three fungi was poor. The fungi grew better when soybean cotyledon fiber was added to DDGS, and cellulase and xylanase activity increased with the increase of soybean fiber content. Porosity was identified as an important factor for SSF because the addition of inert soybean hull alone improved the fungi growth significantly. These data suggest that the nutritional value of DDGS and soybean cotyledon fiber as monogastric animal feed could be greatly enhanced by SSF treatment.     

Xylanase Isozymes from the Newly Isolated Bacillus sp. CKBx1D and Optimization of Its Deinking Potentiality

Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480?U/ml) at 36?h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94?kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2?h of continuous shaking at constant temperature of 35?°C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.     

Study on the production of a xylanolytic complex frompenicillium canescens 10-10c

We screened about a hundred microorganisms (including unidentified yeasts, fungi, and bacteria) for their ability to produce xylanolytic enzymes. About 40 of them were hemicellulolytic; among these, we selectedPenicillium canescens 10- 10c for detailed study because of its ability to produce an interesting enzymatic complex in quantity. The xylanase complex was cellulase-free, and had an optimal activity at pH 4.6–5.0 and 55–60?C on birchwood xylan. The best production was on soya meal and wheat straw; expression of the xylanase was repressed by glucose, xylose, and lactose. The optimization of culture medium and mode (fed-batch) enabled us to improve the production three to four times. The importance of the mixing conditions on the biomass and xylanase production is also reported.     

Effect of Microwave Irradiation on Xylanase Production from Wheat Bran and Biobleaching of Eucalyptus Kraft Pulp

Microwave irradiation (MWI) was used as pretreatment of wheat bran and eucalyptus kraft pulp to examine its effect on xylanase production by Bacillus halodurans FNP 135 using solid state fermentation and biobleaching with xylanase, respectively. Irradiation of wheat bran under optimized conditions (600?W, 6?min, and 20?% consistency) resulted in 56.8, and 31.7?% increase in xylanase yield and water absorbance of wheat bran and 17.3?% reduction in reducing sugars content. Optimized MWI of kraft pulp at 850?W, 2?min, and 20?% consistency led to 0.9?% increase in brightness, 10?% decrease in kappa number, 7.7?% increase in water absorbance, 4.6?% decrease in tear factor, 0.9?% increase in burst factor, and 7.5?% increase in viscosity. Also, MWI enhanced xylanase-mediated biobleaching by increasing brightness (1.1?%) and decreasing kappa number (14.3?%) and leading to a total of about 20?% reduction in chlorine consumption. MWI is an economical, efficient, and environment-friendly pretreatment of wheat bran and pulp for enhanced enzyme yield and rapid heating, respectively.     

Aspergillus fumigatus Thermophilic and Acidophilic Endoglucanases

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer’s spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 °C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L^-1 and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 °C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g^-1 of dry substrate (wheat bran and sugarcane bagasse) within 3 days.