Crude Xylanase Research Articles

Potential of Laceyella sacchari strain B42 crude xylanase in biobleaching of kraft pulp

Xylanase producing thermophilic actinomycetes strain B42 was isolated from bagasse. This strain was enriched on oat spelt xylan agar medium and screened onto xylan-congo red agar plate by the xylanolysis method. The phylogenetic analysis using 16S rDNA sequence data confirmed that strain B42 showed highest homology (99.0%) with Laceyella sacchari and was identified as Laceyella sacchari strain B42. Maximal xylanase production was achieved at the incubation period of 48 h with the xylanase and cellulase activities as 24.5 and 0.08 U/ml, respectively. The optimum pH and optimum temperature of L. sacchari strain B42 xylanase was found to be 9.0 and 60°C, respectively. Xylanase was thermostable at 60°C for 1 h and retained 90% of its activity up to 6 h at this temperature, and subsequently enzyme retained 75 and 60% activity at 70 to 80°C, respectively after 6 h. At biobleaching of kraft pulp, enzyme released sufficient amount of phenolic and hydrophobic compounds. The ultraviolet (UV absorption spectrum of the compounds released by enzyme treatment indicated the presence of lignin in the released coloring matter. The enzymatic biobleaching of kraft pulp caused ~12% reduction of kappa number, 6.67 fold releases of reducing sugars and 10% decrease of lignin content at xylanase optimum dose (60 U/g) and time (4 h). Keywords : Biobleaching, kappa number, pulp and paper industry, thermostable, xylanase, 16S rDNA African Journal of Biotechnology Vol. 12(6), pp. 570-579

Characterization of a NewProvidenciasp. Strain X1 Producing Multiple Xylanases on Wheat Bran

Providencia sp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications.

A Box–Behnken design approach for the production of xylanase by Aspergillus candidus under solid state fermentation and its application in saccharification of agro residues and Parthenium hysterophorus L.

Xylanase production by indigenous Aspergillus candidus using wheat bran as a substrate under solid state fermentation was studied. Inorganic nitrogen source ammonium nitrate was found best among all the nitrogen sources tested. Physical parameters like time of incubation, temperature and moisture content were optimized using Box–Behnken design and under optimized condition maximum xylanase activity found 770U/gds. Low amount of CMCase (77U/gds) and FPase (26.12U/gds) were produced concomitantly with xylanase. The crude xylanase preparation was used for saccharification of aqueous ammonia treated of various lignocellulosic materials like corn cob, wheat straw, sugar cane bagasse and Parthenium weed. Corn cob produced maximum sugar yield and further response surface method was employed to optimized saccharification yield. Under optimized condition maximum reducing sugar yield were found to be 438.47mg/g which was found significantly high considering earlier studies available in the literature.

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70-650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.

Characterization of a Recombinant Thermostable Xylanase from Hot Spring Thermophilic Geobacillus sp. TC-W7

A xylanase-producing thermophilic strain, Geobacillus sp. TC-W7, was isolated from a hot spring in Yongtai (Fuzhou, China). Subsequently, the xylanase gene that encoded 407 amino acids was cloned and expressed. The recombinant xylanase was purified by GST affinity chromatography and exhibited maximum activity at 75 degrees C and a pH of 8.2. The enzyme was active up to 95 degrees C and showed activity over a wide pH range of 5.2 to 10.2. Additionally, the recombinant xylanase showed high thermostability and pH stability. More than 85% of the enzyme's activity was retained after incubation at 70 degrees C for 90 min at a pH of 8.2. The activity of the recombinant xylanase was enhanced by treatment with 10 mM enzyme inhibitors (DDT, Tween-20, 2-Me, or TritonX-100) and was inhibited by EDTA or PMSF. Its functionality was stable in the presence of Li+, Na+, and K+, but inhibited by Hg2+, Ni2+, Co2+, Cu2+, Zn2+, Pb2+, Fe3+, and Al3+. The functionality of the crude xylanase had similar properties to the recombinant xylanase except for when it was treated with Al2+ or Fe2+. The enzyme might be a promising candidate for various industrial applications such as the biofuel, food, and paper and pulp industries.

Production, Purification, and Characterization of a Xylooligosaccharides-forming Xylanase from High-butanol-producing Strain Clostridium sp. BOH3

An endo-acting xylanase is isolated from the culture medium of Clostridium sp. BOH3 when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) is used as a carbon source. Crude xylanase is purified by using an anionic Q-column with a yield of 39 %. The pure xylanase has a molecular weight of 35.8 kDa, and it shows optimal activity at pH 5 and 60 °C. When beechwood xylan is used as a substrate, this xylanase liberates short oligosaccharides (XOS) predominantly, ranging from xylobiose (X2) to xylopentaose (X5). However, no xylose can be detected, suggesting that this is an endo-β-1,4-xylanase. Kinetic study of this xylanase reveals that Km and Vmax are 1.36 mg/ml and 212 μmol/(min. mg protein), respectively. On the basis of amino acid sequence, this enzyme shows homology to xylanase (xynb) from Clostridium acetobutylicum ATCC 824, but this enzyme has several distinctive characteristics. For example, its activity can be enhanced with the addition of divalent metal ions, and it produces XOS exclusively when xylan is used as a substrate. These unique characteristics suggest that this is a new enzyme.

Structural features and antioxidant activity of xylooligosaccharides enzymatically produced from sugarcane bagasse

Xylooligosaccharides (XOS) were prepared from xylan-rich hemicelluloses isolated by potassium hydroxide from sugarcane bagasse by hydrolysis with crude xylanase secreted by Pichia stipitis. Hydrolysis for 12h produced XOS with a maximum yield of 31.8%, equivalent to 5.29mgmL−1 in the hydrolyzate. XOS with degrees of polymerization (DP) from 2 to 4 (xylobiose, xylotriose, and xylotetraose) were the major components in the hydrolysates, whereas the oligosaccharides with higher DP of 5–6 (xylopentaose and xylohexose) showed a constant low level. FT-IR and NMR (1H, 13C, HSQC) demonstrated that XOS contained Araf and 4-O-Me-α-d-GlcpA residues. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay showed that the XOS exhibited concentration-dependent antioxidant activity. The results obtained indicate that the XOS produced from sugarcane bagasse can be employed in food-related applications.

ECF AND TCF BLEACHING OF SACCHARUM OFFICINERUM-CO89003 BAGASSE SODA-AQ PULP WITH ALKALI-THERMO-TOLERANT CRUDE XYLANASE FROM COPRINELLUS DISSEMINATUS SW-1 NTCC1165

An alkali-thermo-tolerant crude xylanase from Coprinellus disseminatus SW-1 NTCC1165 produced under solid-state fermentation conditions improves the brightness of sugarcane bagasse soda-AQ pulp by 7.3, 4.7, 6.1, and 8.2% in XODED, XOD(EOP)DP, OX(EOP)P, and XO(EOP)P bleaching sequences, respectively, at an enzyme dose of 8IU/g, a reaction time of 120 min, a consistency of 10%, and a pH of 6.4 at 55 oC. An improvement in brightness by 2.1% for pulp bleached by XO(EOP)P compared to OX(EOP)P sequence validates that xylanase treatment is more effective for hydrolysing lignin-carbohydrates complexes before oxygen treatment. AOX after XODED and XOD(EOP)DP sequences is reduced by 41.43 and 40%, respectively, compared to controls, but an increase in COD and color in studied bleaching sequences is attributable to the hydrolysis of hemicelluloses and the release of lignin-carbohydrates complexes after xylanase treatment. Xylanase treatment modifies fibre surface by introducing cracks, peelings, swelling, and external fibrillation, which facilitates faster penetration of bleach chemicals by disrupting physical barriers, as revealed by scanning electron microscopy.

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL(-1), respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k (cat)/K (m) = 1,440mg mL(-1) s(-1)) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117% immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8% higher production of xylooligosaccharides compared to free enzyme. After 17cycles, the immobilized enzyme retained 92% of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Production and characterization of acidophilic xylanolytic enzymes from Penicillium oxalicum GZ-2

Multiple acidophilic xylanolytic enzymes were produced by Penicillium oxalicum GZ-2 during growth on wheat straw, rice straw, corn stover, and wheat bran. The expression of xylanase isoforms was dependent on substrate type and nitrogen source. The zymograms produced by the SDS–PAGE resolution of the crude enzymes indicated that wheat straw was the best inducer, resulting in the highest xylanase (115.2U/mL) and β-xylosidase (89mU/mL) activities during submerged fermentation. The optimum temperature and pH for xylanase activity were 50°C and 4.0, respectively; however, the crude xylanase enzymes exhibited remarkable stability over a broad pH range and showed more than 90% activity at 50°C for 30min at pH 4.0–8.0. The results revealed that P. oxalicum GZ-2 is a promising acidophilic xylanase-producing microorganism that has great potential to be used in biofuels, animal feed, and food industry applications.

Improved Purity and Immunostimulatory Activity of β-(1→3)(1→6)-Glucan from Pleurotus sajor-caju Using Cell Wall-Degrading Enzymes

The objective of this work was to improve the purity of β-(1→3)(1→6)-glucan in the native triple helical structure from the fruiting bodies of Pleurotus sajor-caju for effective biological function using cell wall-degrading enzymes. A crude carbohydrate was extracted with hot water, then treated with crude xylanase and cellulase from Paenibacillus curdlanolyticus B-6. β-Glucan in the extract was purified to homogeneity with a single and symmetrical peak using 650M DEAE Toyopearl and Sepharose CL-6B column chromatography. The purity of β-glucan was confirmed by high-performance size-exclusion chromatography. Purified β-glucan was obtained at a purity of up to 90.2%. The Congo red reaction and atomic force microscopy indicated that the purified β-glucan exhibited a triple helix conformation. Purified β-glucan was able to effectively up-regulate the functions of macrophages such as nitric oxide (NO) and tumor necrosis factor (TNF-α) production.

Assessment of a Thermostable Xylanase from Paenibacillus sp. ASCD2 for Application in Prebleaching of Eucalyptus Kraft Pulp

Cellulase free thermostable xylanase from Paenibacillus sp. ASCD2 was used to evaluate its applicability in prebleaching of eucalyptus kraft pulp. Pretreatment of eucalyptus kraft pulp with crude xylanase (40 U per gram of dry pulp) at 60 °C for 3 h showed 21.8 ± 0.04 % reduction in kappa number of kraft pulp along with release of 17.4 ± 0.36 mg/g reducing sugars. Increase in the absorbance at 237 and 465 nm during biobleaching of kraft pulp indicated release of chromophores and hydrophobic compounds from the pulp fibres respectively. Scanning electron microscopic studies indicated that there was increase in opening of the microfibres, swelling, separation and loss in packness of the pulp fibres of xylanase pretreated chemically bleached pulp. The results of the present study proved that xylanase treatment prior to chemical bleaching can reduce the use of chemicals for bleaching of pulp in paper pulp industry.

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

Comparison of the Kinetic Properties of Crude and Purified Xylanase from <i>Bacillus pumilus</i> with Commercial Xylanase from <i>Aspergillus niger</i>

Xylanases showing better stability at alkaline pH and higher temperatures have potential applications in several industrial processes. The objective of this study is to compare the kinetic properties and stability of crude and purified xylanase from Bacillus pumilus with commercial xylanase from Aspergillus niger . Bacillus pumilus which can grow and produce xylanase above 40oC and pH 9.0 was selected for this study. Crude, purified and commercial xylanases showed zero order kinetics for 10 minutes and highest activity was obtained at 60°C and pH 9.0. The optimum pH for crude, purified and commercial xylanases were 9.0, 9.0 and 7.0 respectively at 60°C and the Michaelis constant by Lineweaver-Burk Plot for xylan were 3.1, 2.3 and 0.03gL -1 under the respective optimized conditions. The half-life of the crude and purified xylanase was highest at pH 9.0 and 50°C while that for the commercial enzyme was at pH 7.0 and at 60°C. Key words - Xylanase; Kinetic properties; Michaelis constant; Vmax; Stability; Half life DOI: http://dx.doi.org/10.4038/vingnanam.v10i1.4072 Ving. Journal of Science 2011 , Vol. 10, No. 1, 1-7

Production of xylanase under solid-state fermentation by Aspergillus tubingensis JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H(2)SO(4)) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.

Identification of xylanase producing Bacillus licheniformis strain C1 and properties of crude xylanase

In the present investigation xylanase producing bacteria was isolated from compost. A total of 95 xylanolytic bacteria were isolated on oat spelt xylan agar medium and screened by the xylanolysis method. Out of these 95 isolates, only one bacterial isolates, strain C1 was selected for further study on the basis of zone of hydrolysis on xylan-congo red agar plate. This strain was identified by 16S rDNA analysis. The phylogenetic analysis using 16S rDNA sequence data showed that isolate C1 showed highest nucleotide identity of 98% with Bacillus licheniformis strain CICC 10181 (GenBank accession no. GQ375235) and identified as Bacillus licheniformis strain C1. Bacillus licheniformis strain C1 was gram positive and rod shaped. Morphology of Bacillus licheniformis strain C1 showed- smooth texture, medium size, opaque transparency, creamish-white colour and serrated margin. Maximal xylanase production for Bacillus licheniformis strain C1 was achieved at the incubation period of 48 h. Xylanase and cellulase activities were determined as 20.0 U/ml and 1.3 U/ml, respectively. The optimum pH and optimum temperature for xylanase activity was found to be 7.0 and 60°C, respectively. Xylanase was found to be thermostable at 60°C for 1h and retained 90% of its activity upto 6 h at this temperature. Approximately, 74% and 70% of its activity was retained at 70°C and 80°C respectively, after 6 h of incubation. All of these properties of the Bacillus licheniformis strain C1 xylanase make the suitability of this enzyme for its use in feed and baking industry.

Bio-conventional bleaching of kadam kraft-AQ pulp by thermo-alkali-tolerant xylanases from two strains of Coprinellus disseminatus for extenuating adsorbable organic halides and improving strength with optical properties and energy conservation

Two novel thermo-alkali-tolerant crude xylanases namely MLK-01 (enzyme-A) and MLK-07 (enzyme-B) from Coprinellus disseminatus mitigated kappa numbers of Anthocephalus cadamba kraft-AQ pulps by 32.5 and 34.38%, improved brightness by 1.5 and 1.6% and viscosity by 5.75 and 6.47% after (A)XE(1) and (B)XE(1)-stages, respectively. The release of reducing sugars and chromophores was the highest during prebleaching of A. cadamba kraft-AQ pulp at enzyme doses of 5 and 10 IU/g, reaction times 90 and 120 min, reaction temperatures 75 and 65°C and consistency 10% for MLK-01 and MLK-07, respectively. MLK-07 was more efficient than MLK01 in terms of producing pulp brightness, improving mechanical strength properties and reducing pollution load. MLK-01 and MLK-07 reduced AOX by 19.51 and 42.77%, respectively at 4% chlorine demands with an increase in COD and colour due to removal of lignin carbohydrates complexes. A. cadamba kraft-AQ pulps treated with xylanases from MLK-01 to MLK-07 and followed by CEHH bleaching at half chlorine demand (2%) showed a drastic reduction in brightness with slight improvement in mechanical strength properties compared to pulp bleached at 4% chlorine demand. MLK-01 reduced AOX, COD and colour by 43.83, 39.03 and 27.71% and MLK-07 by 38.34, 40.48 and 30.77%, respectively at half chlorine demand compared to full chlorine demand (4%). pH variation during prebleaching of A. cadamba kraft-AQ pulps with strains MLK-01 and MLK-07 followed by CEHH bleaching sequences showed a decrease in pulp brightness, AOX, COD and colour with an increase in mechanical strength properties, pulp viscosity and PFI revolutions to get a beating level of 35 ± 1 °SR at full chlorine demand.

Environmentally friendly totally chlorine free bleaching of wheat straw pulp using novel cellulase-poor xylanases of wild strains of Coprinellus disseminatus

Cellulase-poor crude xylanases of Coprinellus disseminatus, strains SH-1 NTCC 1163 (enzyme-A) and SH-2 NTCC 1164 (enzyme-B) produced under optimum conditions of solid-state fermentation (SSF), were used in bio-bleaching of wheat straw soda-AQ pulp in totally chlorine free (TCF) bleaching sequences. Kappa number reductions of 56% and 58% with respect to oxygen-delignified pulps were obtained after the sequences OXAE and OXBE, respectively. Significant increases in pulp brightness of 6.07% (enzyme-A) and 3.34% (enzyme-B) with slight decreases in some strength properties (due to the removal of hemicelluloses) were observed. Removal of hemicelluloses was further validated by an increase in pulp viscosity (6.07%, and 4.58%), COD (40%, and 38%), and facilitation of lignin removal, as indicated by colour values (48% and 45%) for OXAEQPP and OXBEQPP bleached pulps, respectively, over the control. Crude xylanases from C. disseminatus SH-1 NTCC 1163 and SH-2 NTCC 1164 can successfully be used for TCF bleaching of pulps owing to their high temperature and pH toleranance, and cellulase-poor nature, thus adding to the search for environment-friendly bleaching solutions for the pulp and paper industry.

High level expression of a recombinant xylanase by Pichia pastoris NC38 in a 5 L fermenter and its efficiency in biobleaching of bagasse pulp

A genetically modified XynA gene from Thermomyces lanuginosus was expressed in Pichia pastoris under the control of GAP promoter. P. pastoris expressed greater levels of xylanase (160IUml−1) on BMGY medium without zeocin after 56h. The xylanase production by recombinant P. pastoris was scaled up in a 5L fermenter containing 1% glycerol and the highest xylanase production of 139IUml−1 was observed after 72h. Further studies carried out in fermenter under controlled pH (5.5) yielded a maximum xylanase production of 177IUml−1 after 72h. The biobleaching efficacy of crude xylanase was also evaluated on bagasse pulp and a brightness of 47.4% was observed with 50IU of crude xylanase used per gram of pulp, which was 2.1 points higher in brightness than the untreated samples. Reducing sugars (24.8mgg−1) and UV absorbing lignin-derived compounds values were considerably higher with xylanase treated samples.

Production of xylanase by Promicromonospora sp MARS with rice straw under non sterile conditions

Quantitative differentiation of the inoculated Promicromonospora sp MARS and indigenous microbial flora of rice straw is described using ERIC-PCR during the production of xylanase in non sterile solid state fermentation (NS-SSF) to reduce the cost of enzyme production. Under non-sterile solid state fermentation with rice straw, Promicromonospora sp MARS produced 85.0 IU/g of xylanase. DNA fingerprints of unknown bacterial isolates obtained on BHA + xylan exactly matched with that of Promicromonospora sp MARS. Using PCR based enumeration; population of Promicromonospora sp MARS was 67.5 × 10 5 cfu/g after 48 h which increased to 23 × 10 6 cfu/g after 96 h which coincided with the maximum xylanase activity as compared to 14 × 10 4 cfu/g in control after 96 h. Under submerged fermentation (SmF), after 48 h, Promicromonospora sp MARS produced maximum xylanase (42.2 IU/ml) using rice straw as substrate followed by 19.9 and 20.4 IU/ml when Promicromonospora sp MARS was grown on rice husk and wheat straw, respectively. Optimum pH and temperature of the crude xylanase were 8.0 and 65 °C. Crude enzyme was 95% stable for 180 min of incubation at pH 8.0 and was stable at 65 °C for 240 min.