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.     

Purification and Characterisation of a Thermostable β-Xylosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> van Tieghem of Potential Application in Lignocellulosic Bioethanol Production

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable β-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70–75 °C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72 h at 60 and 65 °C, respectively, with 52% residual activity detected after 18 h at 70 °C. Available data indicates that the purified β-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-β-d-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl β-d-glucopyranoside and p-nitrophenyl α-l-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-β-xylanase in the hydrolysis of beechwood xylan at 65 °C. During hydrolysis of pretreated straw lignocellulose at 70 °C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6 h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable β-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.     

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant,Thermostable β-Xylosidase from a Tropical Strain of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> CBS 135684

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60–70 °C) over a pH range of 5–9 with a specific activity of 163.3 units (U) mg^?1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K _i value of 18 mM. The K _m and V _max values against p-nitrophenyl-β-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 μmol min^?1 mg^?1 protein, respectively. When combining this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g^?1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g^?1 β-xylosidase and 16 U g^?1 xylanase after incubation for 4 h at 70 °C and pH 6.0.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M _r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k _cat/K _M) of 0.0048?±?0.0010 s^?1 mM^?1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.     

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 %.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

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.     

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.     

Isolation and Cultivation of a Xylanolytic Bacillus subtilis Extracted from the Gut of the Termite Reticulitermes santonensis

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller’s grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller’s grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8.     

Some properties of a thermostable β-xylosidase fromRhodothermus marinus

The extracellular β-xylosidase (EC 3.2.1.37) excreted by the thermophilic eubacteriumRhodothermus marinus when grown on xylan has been investigated. The enzyme has been partially purified by ultrafiltration and gel filtration, and some of its characteristics are presented.Rhodothermus marinus grew on xylan with μmax= 0.4 h^? and the α-xylosidase activity was 50 nkat/mL after 24 h in a batch fermentation. The α-xylosidase activity had a half-life of more than 1 h at 90°C and of 14 h at 85 °C. At 80°C, 80% of the initial activity remained after 24 h. The initial activity increased with increasing temperature, showing maximal activity at 90°C. The β-xylosidase had a pH-optimum of 6 and was stable in the range between pH 5 and 9. At pH 10 and 11, 82 and 66%, respectively, of the initial activity remained after 24 h when incubated at 65°C. The molecular weight was estimated to be 169,000 dalton by gelfiltration.     

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.     

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 β-Xylosidase II from Caulobacter crescentus

In the present work, the gene xynB2, encoding a ??-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 ??-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?C7.5 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???mol?min^?1 for ??-nitrophenyl-??-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 ??-xylosidases have been characterized, this work provides a good contribution to this group of enzymes.     

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.     

Use of an (Hemi) Cellulolytic Enzymatic Extract Produced by Aspergilli Species Consortium in the Saccharification of Biomass Sorghum

This study evaluated the production of lignocellulose-degrading enzymes by solid-state fermentation (SSF) using a microbial consortium of Aspergillus fumigatus SCBM6 and A. niger SCBM1 (AFN extract). The fungal strains were cultivated in sugarcane bagasse (SCB) and wheat bran (WB) as lignocellulosic substrates for 7 days at 30 °C. After SSF, the highest peaks of enzyme production were 150 and 80 U g⁻¹ for β-xylosidase and β-glucosidase at 48 h, 375 U g⁻¹ for xylanase at 96 h, and 80 U g⁻¹ for endoglucanase and 4 U g⁻¹ for cellulase activity on filter paper (FPase) at 144 h. The efficiency of the produced AFN extract was investigated in the enzymatic hydrolysis of crude biomass sorghum (BS) and after the removal of extractives (ES). After saccharification, the glucose and xylose concentrations were 10× superior in ES than in BS hydrolysate (2.5 g L⁻¹ after 12 h). The presence of inhibitors of alcoholic fermentation, such as formic acid, was also reduced in ES hydrolysates, indicating that the removal of extractives positively contributed to the effectiveness of enzymatic hydrolysis of biomass sorghum using AFN extract.

     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4?kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40?°C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5?mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-??-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24?mg?ml^?1 and 58.21???mol?min^?1?mg^?1 protein, respectively.     

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

Proteases are some of the most important industrial enzymes, and one of their main applications is for the production of cheese in the dairy industry. Due to a shortage of animal rennet, microbial coagulant proteases are being sought. In this work, the production of microbial rennet from Thermomucor indicae-seudaticae N31 was studied in submerged fermentation. The best enzyme production was obtained in a fermentation medium containing 4 % wheat bran as the substrate in 0.3 % saline solution, incubated for 72 h at 45 °C and 150 rpm. The value of the milk clotting activity (MCA) was 60.5 U/mL, and the ratio to proteolytic activity (MCA/PA) was 510. The crude enzyme showed optimum pH at 5.5 and two peaks of optimum temperature (MCA at 65 °C and PA at 60 °C). The MCA was stable in the pH range 4.0–4.5 for 24 h and up to 55 °C for 1 h. It was stable during storage at different temperatures (?20 to 25 °C) for 10 weeks. Based on these results, we conclude that microbial rennet from T. indicae-seudaticae N31 produced by submerged fermentation showed good prospects of replacing traditional rennet.