Crude Xylanase Research Articles

Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa

Xylooligosaccharides (XOS) with DP 2–4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25Ug−1 substrate, pH 5.4 and 50°C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated.

BIO-CONVENTIONAL BLEACHING OF KRAFT-AQ PULP OF A. CADAMBA BY CRUDE XYLANASES FROM COPRINELLUS DISSEMINATUS MLK-03 AND EFFECT OF RESIDUAL ENZYME ON EFFLUENT LOAD

A new thermo-alkali-tolerant crude xylanase from Coprinellus disseminatus decreased kappa number by 34.38% and improved brightness and viscosity by 1.6 and 6.47% respectively after XE1-stage during prebleaching of Anthocephalus cadamba kraft-AQ pulp. At 2.4% chlorine demand, crude xylanase in a XECEHH (X= enzymatic prebleaching stage, E= extraction stage, C= chlorination stage, H= hypochlorite stage) bleaching sequence improved pulp brightness, tensile index, burst index, and double fold numbers by 3.66%, 4.78%, 6.38%, and 11.11%, respectively with a reduction in viscosity (10.59%) and tear index (10.77%) compared to the control. Combined bleach effluent of the XECEHH sequence mitigated adsorable organic halides (AOX) by 21% and increased chemical oxygen demand (COD), bio-chemical oxygen demand (BOD), and colour by 67.18%, 84.78%, and 97.53%, respectively, compared to the control. Residual enzymes that entered during enzymatic prebleaching stage decreased AOX, COD, BOD, and colour of combined effluent of the XECEHH bleaching sequence progressively and on 6th day, and these were reduced by 23.78%, 0.04%, 15.00%, and 0.61%, respectively, compared to the control.

Bio-conventional bleaching of kraft-AQ pulp of A. cadamba by crude xylanases from Coprinellus disseminatus MLK-03 and effect of residual enzyme on effluent load

A new thermo-alkali-tolerant crude xylanase from Coprinellus disseminatus decreased kappa number by 34.38% and improved brightness and viscosity by 1.6 and 6.47% respectively after XE1-stage during prebleaching of Anthocephalus cadamba kraft-AQ pulp. At 2.4% chlorine demand, crude xylanase in a XECEHH (X= enzymatic prebleaching stage, E= extraction stage, C= chlorination stage, H= hypochlorite stage) bleaching sequence improved pulp brightness, tensile index, burst index, and double fold numbers by 3.66%, 4.78%, 6.38%, and 11.11%, respectively with a reduction in viscosity (10.59%) and tear index (10.77%) compared to the control. Combined bleach effluent of the XECEHH sequence mitigated adsorable organic halides (AOX) by 21% and increased chemical oxygen demand (COD), bio-chemical oxygen demand (BOD), and colour by 67.18%, 84.78%, and 97.53%, respectively, compared to the control. Residual enzymes that entered during enzymatic prebleaching stage decreased AOX, COD, BOD, and colour of combined effluent of the XECEHH bleaching sequence progressively and on 6th day, and these were reduced by 23.78%, 0.04%, 15.00%, and 0.61%, respectively, compared to the control.

Identification of Paenibacillus sp. 2S-6 and application of its xylanase on biobleaching

A Paenibacillus sp. strain 2S-6 was isolated from the black liquor of the first brownstock washing stage of kraft pulping process and identified by its 16S rDNA sequence. This bacterial strain utilized a variety of saccharides and polysaccharides as carbon source, but neither lignin nor lipids. Crude xylanase from Paenibacillus sp. 2S-6 was produced in a 5 L laboratory fermenter at 37 °C, pH 7. After 24 h, up to 10.5 IU xylanase per mg of protein in the crude extract of fermentation broth was obtained. After two-stage ultrafiltration, the optimal activity of partially purified xylanase reached 60.51 IU/mg at 50 °C, pH 6. A major band indicating molecular weight of 33 kDa was shown on SDS-PAGE for the partially purified xylanase. After 4 h at 60 °C, 48.99% and 31.25% residual xylanase activities were demonstrated at pH 7 and 9, respectively. Efficacy of its xylanase on the bleaching agent saving was demonstrated by using 5 IU xylanase per gram oven-dried pulp prior to bleaching, referred as biobleaching. Identical levels of brightness and higher levels of viscosity were obtained for the xylanase pretreated eucalypt kraft pulps followed by a 20% reduction of the bleaching agent dosage in the first step of a commercial C70/D30-Eo-D bleaching sequence.

Purification, characterization and mass spectroscopic analysis of thermo-alkalotolerant β-1, 4 endoxylanase from Bacillus sp. and its potential for dye decolorization

A Bacillus sp., isolated from sludge and sediments of pulp and paper mill, was found to produce xylanase in a synthetic culture media containing oat spelt xylan (1% w/v) and 10% black liquor as inducers along with 2.5% (w/v) sucrose as additional carbon source. The purified enzyme was highly thermostable with half-life of 10min at 90°C and pH 8. The enzyme was stable over a broad range of pH (pH 6–10) and showed good thermal stability when incubated at 70°C. Chemicals like EDTA, Hg2+, Cu2+ and solvents like glycerol and acetonitrile completely inhibited enzyme activity at high concentration. The molecular weights of the purified enzyme, determined by matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF/MS) analysis was analogous to the results obtained from SDS-PAGE, i.e. 55kDa. Kinetic parameters were determined by using oat spelt xylan as substrate. The KM and Vmax values of the enzyme were 4.4mg/ml and 287U/mg respectively. At high xylan concentrations (>70mg/ml) a substrate inhibition phenomenon of the enzyme was observed. In addition, crude xylanase showed enormous potential for decolorization of various recalcitrant dyes.

Enzymatic bleaching of kraft pulp by xylanase from Aspergillus sydowii SBS 45

Crude xylanase from Aspergillus sydowii SBS 45 was tested for enzymatic bleaching of kraft (Decker) pulp. After optimization of three parameters, consistency of pulp, retention time and enzyme dose, considerable increase in the release of UV and visible absorbance spectra of materials and reducing sugars was observed, which clearly indicated the action of xylanase on pulp. Final brightness of pulp was increased from 29.42 to 70.42% and kappa number was reduced from 15.93 to 1.61, when 25 U of xylanase was given with a retention time of 5 h and at a consistency of 10%. When 10 U g(-1) xylanase was given, 14.3% elemental chlorine and 14.3% H(2)O(2) could be reduced and when 25 U g(-1) xylanase was given 14.3% elemental chlorine and 28.6% H (2)O(2) could be reduced thereby retaining the brightness at control level.

Production of Crude Xylanase from Thermoascus Aurantiacus CBMAI 756 Aiming the Baking Process

In recent years, the baking industry has focused its attention on substituting several chemical compounds with enzymes. Enzymes that hydrolyze nonstarch polysaccharides, such as xylanase, lead to the improvement of rheological properties of dough, loaf specific volume, and crumb firmness. The purpose of this study was to find a better solid-state fermentation substrate to produce high levels of xylanase and low levels of protease and amylase, which are enzymes involved in bread quality, from Thermoascus aurantiacus CBMAI 756. Wheat bran, corncob, and corn straw were used as energy sources. The enzyme extract of corncob showed high xylanase activity (130 U/mL) and low amylase and protease activity (<1 and 15 U/mL, respectively). This enzyme profile may be more profitable for the baking industry, because it results in a slower degradation of gluten. Our results confirm this finding, because the enzyme obtained by fermentation in corncob resulted in a gluten with a higher specific volume than all the other substrates that were tested. The crude xylanase presented maximum activity at a pH of 5, and the optimum temperature was 75 °C. It was stable up to 70 °C for an hour and at a pH range from 4 to 10.

Bleach-boosting effect of crude xylanase from Bacillus stearothermophilus SDX on wheat straw pulp

Pretreatment of wheat straw pulp using cellulase-free xylanase produced from Bacillus stearothermophilus SDX at 60°C for 120min resulted in 4.75% and 22.31% increase in brightness and whiteness, respectively. Enzyme dose of 10U/g of oven dried pulp at pH 9 decreased the kappa number and permanganate number by 7.14% and 5.31%, respectively. Further chlorine dioxide and alkaline bleaching sequences (CDED(1)D(2)) resulted in 1.76% and 3.63% increase in brightness and whiteness, respectively. Enzymatic prebleaching of pulp decreased 20% of chlorine consumption without any decrease in brightness. Improvement in various pulp properties like viscosity, burst factor, burstness, breaking length, double fold, gurley porosity, tear factor, and tearness were also observed after bleaching of xylanase treated wheat straw pulp.

Site-Directed Mutagenesis and Thermostability of Xylanase XYNB from Aspergillus niger 400264

Xylanase is one of the most important hemicellulases in industry. However, its low thermostability limits its applications. In this study, one thermostable xylanase-producing strain 400264 was obtained from screening 11 Aspergillus niger strains (producing thermotolerant xylanase), and the optimum temperature of crude xylanase extracted from it was 55°C. Original activity of the crude xylanase is 64% at 60°C and 55% at 85°C with an incubation time of 30 min, respectively. After the expression of recombinant xylanase gene (xynA/xynB), the XYNB (xylanase B) showed higher thermostability than XYNA (xylanase A). Recombinant enzyme XYNB retained 94% of its activity for 10 min at 85°C, while XYNA with no activity left. Site-directed mutagenesis was performed to replace Ala33 of XYNB by Ser33 resulting 19% decrease in enzyme activity after incubating at 85°C for 30 min. It suggested that the Ala33 residue may have a certain effect on the thermophilic adaptation of xylanase.

Bio-conventional bleaching of wheat straw soda–AQ pulp with crude xylanases from SH-1 NTCC-1163 and SH-2 NTCC-1164 strains of Coprinellus disseminatus to mitigate AOX generation

Two novel cellulase-poor xylanases from Coprinellus disseminatus SH-1 NTCC-1163 (enzyme-A) and SH-2 NTCC-1164 (enzyme-B) produced under solid-state fermentation mitigated kappa number of wheat straw soda-AQ pulps by 24.38 and 27.94% respectively after XE stages. The release of reducing sugars and chromophores was highest for both the enzymes at 10IU/g and reaction time 180min for 55°C at variable consistencies that is, 10% for enzyme-A and 5% for enzyme-B. (A)XECEHH and (B)XECEHH sequences improved brightness by 5.17 and 2.58% respectively at 4.5% chlorine charge. AOX in (A)XECEHH and (B)XECEHH sequences reduced by 56.11 and 55.75% respectively at 4.5% chlorine charge and 68.34 and 67.98% respectively at 2.25% chlorine charge respectively compared to control. Both the enzymes showed improvement in double fold and tear index with a decrease in burst and tensile index. SEM showed peeling, cracking and delamination in fibers due to enzyme treatment thus facilitating the penetration of bleach chemicals.

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.

Thermoactive cellulase-free xylanase production from alkaliphilic Bacillus strains using various agro-residues and their potential in biobleaching of kraft pulp

The four bacterial strains were isolated on media containing xylan and screened for xylanase activity. The bacterial strains (Ag12, Ag13, Ag20 and Ag32) were characterized based on morphological, biochemical and physiological characters and identified as belonging to the genus Bacillus. The effects of different factors such as pH (7.0 – 10.0), temperature (25.0 – 50.0°C) and inexpensive agro-residues (wheat straw, wheat bran and corncob) on xylanase production of strains were studied under shake flask conditions. Maximal enzyme activities were obtained by cultivation in birch-wood xylan, but high enzyme production was also obtained on wheat straw and corncob when cultivated at pH 8.5. Under optimized fermentation conditions, no cellulolytic activity were detected on the crude extracts. The effects of temperature (40.0 – 80.0°C), pH (6.0 – 10.0) and salt concentration (1.0, 5.0 and 10.0%) on the xylanases activity were determined. The maximum activity was obtained temperature 60.0°C and pH at 9.0. The enzyme was stable at 60.0°C for more than 60 min, suggesting that the xylanases of Bacillus strains are thermoactive and being of interests for biobleaching processes. The effectiveness of crude xylanases from the strains Ag12, Ag20 and Ag32 on kraft pulp were carried out at pH 9.0 at 60.0°C. Biobleaching studies of kraft pulp with xylanases and its subsequent treatment with 1.0% EDTA (30 min at 50.0°C) and peroxide (80 min at 70.0°C), showed that the enzymes reduced the kappa number by 27.4, 61.7 and 75.3% and enhanced the brightness by 1.0, 1.5 and 3.0% from xylanases produced by strains Ag12, Ag20 and Ag32, respectively. These results suggest that the application of this xylanases to the paper and pulp industry may be very promising.

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 beta-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 degrees 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 55 and 60 degrees C for bacterial and fungal xylanase, respectively, and they were thermally stable up to 50 degrees C.

Enhanced production of cellulase-free xylanase by alkalophilic Bacillus subtilis ASH and its application in biobleaching of kraft pulp

This paper reports high level production of a cellulase-free xylanase using wheat bran, a cost-effective substrate, under submerged fermentation by alkalophilic Bacillus subtilis ASH. Production of xylanase was observed even at alkaline pH up to 11.0 and temperature 60 °C, although the highest enzyme titer was recorded at neutral pH and 37 °C. The enzyme production under optimized fermentation was 1.5-fold greater than under unoptimized conditions. Pre-treatment of unbleached pulp of 10% consistency with crude xylanase (6 IU/g o.d. pulp) at 60 ºC for 2 h increased the final brightness by 4.9%. The enzyme treatment reduced the chlorine consumption by 28.6% with the same brightness as in the control. A reduction in kappa number and increase in viscosity was observed after enzyme pre-treatment. Scanning electron microscopy revealed loosening and swelling of pulp fibers. The strength properties viz. grammage, fiber thickness, beating degree, tensile index, breaking length, tear index and double fold of the treated pulp were improved as compared to the control pulp. This study reveals the potentialof B. subtilis ASH xylanase as a biobleaching agent for the paper and pulp industry.

Characterization and Purification a Specific Xylanase Showing Arabinofuranosidase Activity from Streptomyces spp. 234P-16

Streptomyces spp 234P-16 producing xylanase was isolated from soil sample from Padang, West Sumatra, Indonesia. Crude enzyme (produced by centrifuging the culture at 14000 rpm for about 5 minutes) and purified xylanase have an optimum condition at pH 5 and 90oC. Crude xylanase have half life time of 4 hours, whereas purified xylanase have half life time of 2.5 hours at 90oC. The molecular mass of purified xylanase was determined to be 42.4 kDa. The Arabinofuranosidase have a Km and Vmax value of 1.98 mg/mL and 523 μmol/minute/mg, respectivelyKey words: xylanase, xylooligosaccharide, arabinofuranosidase, Streptomyces.

Production of β-xylanase by a Thermomyces lanuginosus MC 134 mutant on corn cobs and its application in biobleaching of bagasse pulp

The production of hemicellulases by Thermomyces lanuginosus SK using oatspelts xylan was examined during submerged cultivation. A high level of extracellular xylanase (346+/-10 U ml(-1)) production was observed on the fifth day; however, accessory enzyme levels were low. T. lanuginosus SK was further subjected to UV and N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. The T. lanuginosus MC 134 mutant showed a 1.5 fold increase in xylanase production on oatspelts xylan, compared to the wild type strain. Xylanase production was further enhanced to 3299+/-95 U ml(-1) by using corn cobs under optimized growth conditions. A reduction in xylanase production was observed in a 5 L fermenter. Also, the biobleaching efficiency of crude xylanase was evaluated on bagasse pulp, and a brightness of 46.07+/-0.05% was observed with the use of 50 U of crude xylanase per gram of pulp. This brightness was 3.6 points higher than that of the untreated samples. Reducing sugars (25.78+/-0.14 mg g(-1)) and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. T. lanuginosus MC 134 has a potential application in the pulp and paper industries.

Hyper production of cellulase-free xylanase by Thermomyces lanuginosus SSBP on bagasse pulp and its application in biobleaching

A cellulase-free xylanase production by Thermomyces lanuginosus SSBP using bagasse pulp was examined under submerged (SmC) and solid-state cultivation (SSC). Higher level of xylanase activity (19,320 +/- 37 U g(-1) dried carbon source) was obtained in SSC cultures than in SmC (1,772 +/- 15 U g(-1) dried carbon source) after 120 h with 10% inoculum. The biobleaching efficacy of crude xylanase was tested on bagasse pulp, and the maximum brightness of 46.1 +/- 0.06% was observed with 50 U of crude xylanase per gram of pulp, which was 3.8 points higher than the brightness of untreated samples. Reducing sugars (26 +/- 0.1 mg g(-1)) and UV-absorbing lignin-derived compounds in the pulp filtrates were observed as maximum in 50 U of crude xylanase-treated samples. T. lanuginosus SSBP has potential applications due to its high productivity of xylanase and its efficiency in pulp bleaching.

CHARACTERIZATION OF STREPTOMYCES sp. 45I-3 XYLANASE

Streptomyces sp 45I-3 producing xylanase was isolated from Kalimantan soil sample. Crude enzyme (produced by centrifuging the culture at 14000 rpm for about 5 minutes) and purified xylanase has an optimum condition at pH 5 and 50 oC. Crude xylanase have half-life time of 2 hours 9 minute at 50 oC, whereas purified xylanase has half-life time of 12 hours. The molecular mass of purified xylanase was determined to be 43.2 and 39.2 kDa.

Biobleaching of banana fibre pulp using Bacillus subtilis C O1 xylanase produced from wheat bran under solid-state cultivation

A cellulase-free xylanase produced by Bacillus subtilis C 01 from wheat bran under solid-state cultivation was tested for its efficacy in biobleaching of raw banana fibre and banana pulp obtained through a mechanical pulping process. Banana pulp samples treated with crude xylanase (450 nkat g(-1) pulp) resulted in a 19.6% increase in the brightness as compared to untreated pulp. The presence of chromophores, hydrophobic compounds and an increased reducing sugar (10.79 mg g(-1) pulp) quantity in the bleached solution after enzymatic treatment indicated the removal of materials that were absorbed at 237 nm from the banana pulp.

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.