Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry

A very high level of cellulase-free, thermostable xylanase has been produced from newly isolated strain of Bacillus pumilus under submerged fermentation in a basal medium supplemented with wheat bran (2%, w/v) pH 8.0 and at 37 °C. After optimization of various production parameters, an increase of nearly 13-fold in xylanase production (5407 IU/ml) was achieved. The produced xylanase is stable in neutral to alkaline pH region at 70 °C. The suitability of this xylanase for use in the bioleaching of eucalyptus Kraft pulp was investigated. A xylanase dose of 5 IU/g of oven dried pulp of 10% consistency exhibited the optimum bleach boosting of the pulp at pH 7.0 and 60 °C after 180 min of treatment. An increase of 5% in brightness along with an increase of 21% and 28% in whiteness and fluorescence respectively, whereas 18% decrease in the yellowness of the biotreated pulp was observed. Enzyme treated pulp when subjected to chemical bleaching, resulted in 20% reduction in chlorine consumption and up to 10% reduction in consumption of chlorine dioxide. Also a reduction of about 16% in kappa number and 83% in permanganate number, along with a reduction in COD value and significant improvement in various pulp properties, viz. viscosity, tensile strength, breaking length, burst factor, burstness, tear factor and tearness were observed in comparison to the conventional chemical bleaching.     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.     

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% H₂O₂ could be reduced and when 25 U g⁻¹ xylanase was given 14.3% elemental chlorine and 28.6% H ₂O₂ could be reduced thereby retaining the brightness at control level.     

Assessment of various commercial enzymes in the bleaching of radiata pine kraft pulps

Four xylanase preparations that are commercially available, namely Cartazyme from Sandoz, Ecopulp from Alko-ICI, Irgazyme from Ciba-Genencor and Pulpzyme HB from Novo Nordisk, were tested in bleaching experiments of kraft pulps from Pinus radiata. The main objective of this study was to optimize a reduction in the consumption of chlorine dioxide in the bleaching sequences C₉₀/D₁₀E_oDED, C₇₀/D₃₀E_oDED and D₁₀₀EDED. Enzymatic treatments led to savings of ClO₂ between 3.5 and 3.9 kg per air-dried tons (ADT) in the three bleaching sequences, without affecting the target brightness of the pulps. In these assays, some minor although reproducible differences in the performance of the enzymes were observed. In most cases, xylanase treatment partially affected the beatability of the pulps, measured as the number of revolutions in the PFI mill required to reach the same tensile index as the respective controls.     

Production of xylanase by Aspergilli using alternative carbon sources: application of the crude extract on cellulose pulp biobleaching

The ability of xylanolytic enzymes produced by Aspergillus fumigatus RP04 and Aspergillus niveus RP05 to promote the biobleaching of cellulose pulp was investigated. Both fungi grew for 4–5 days in liquid medium at 40°C, under static conditions. Xylanase production was tested using different carbon sources, including some types of xylans. A. fumigatus produced high levels of xylanase on agricultural residues (corncob or wheat bran), whereas A. niveus produced more xylanase on birchwood xylan. The optimum temperature of the xylanases from A. fumigatus and A. niveus was around 60–70°C. The enzymes were stable for 30 min at 60°C, maintaining 95–98% of the initial activity. After 1 h at this temperature, the xylanase from A. niveus still retained 85% of initial activity, while the xylanase from A. fumigatus was only 40% active. The pH optimum of the xylanases was acidic (4.5–5.5). The pH stability for the xylanase from A. fumigatus was higher at pH 6.0–8.0, while the enzyme from A. niveus was more stable at pH 4.5–6.5. Crude enzymatic extracts were used to clarify cellulose pulp and the best result was obtained with the A. niveus preparation, showing kappa efficiency around 39.6% as compared to only 11.7% for that of A. fumigatus.     

Xylanase and Mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase and mannanase, singly or in combination, either sequentially or simultaneously. Enzymes were obtained from Streptomyces galbus NR that had been cultivated in a medium, containing either xylan of sugar cane bagasse or galactomannan of palm-seeds, when they were used as sole carbon sources from local wastes in fermentation media. No cellulase activity was detected. Incubation period, temperature, initial pH values and nature of nutritive constituents were investigated. Optimum production of both enzymes was achieved after 5 days incubation on a rotary shaker (200 rpm) at 35 degrees C and initial pH 7.0. Partial purification of xylanase and mannanase in the cultures supernatant were achieved by salting out at 40-60 and 60-80% ammonium sulphate saturation with a purification of 9.63- and 8.71-fold and 68.80 and 62.79% recovery, respectively. The xylanase and mannanase from S. galbus NR have optimal activity at 50 and 40 degrees C, respectively. Both enzymes were stable at a temperature up to 50 degrees C. Xylanase and mannanase showed highest activity at pH 6.5 and were stable from 5.0 to 8.0 and from 5.5 to 7.5, respectively. The partial purified enzymes preparations of xylanase and mannanase enzymes showed high bleaching activity, which is an important consideration for industry. Xylanase was found to be more effective for paper-bleaching than mannanase. When xylanase and mannanase were dosed together (simultaneously), both enzymes were able to enhance the liberation of reducing sugars and improve pulp bleachability, possibly as a result of nearly additive interactions. The simultaneous addition of both enzymes was more effective in pulp treatment than their sequential addition.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Effects of surfactants on the enzymatic bleaching of kraft pulp by xylanase

A xylanase was purified from a commercial crude xylanase, Pulpzyme HC, and used for the bleaching of kraft pulp in the absence or in the presence of nonionic surfactants, Tween 20, Tween 80, and Igepal C930. The purified xylanase has a molecular weight of 23,500 as determined by a reducing SDS-PAGE. Tween 20 was most effective to enhance the efficiency of the enzymatic bleaching of kraft pulp by xylanase.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

‘Single lay out’ and ‘mixed lay out’ enzymatic processes for bio-bleaching of kraft pulp

Xylanase alone as ‘single lay out’ (strategy I) and in combination with pectinase as ‘mixed lay out’ (strategy II) was used to investigate their bio-bleaching potentials. Strategy I was carried at 70 °C using 5 U/g of xylanase at pH 9.5 and 12.5 whereas strategy II was carried out at 70 °C using 5 U/g of each of the enzyme, respectively at pH 9.5. Bio-bleaching caused 15% and 20% less Cl₂ consumption though strategy I and II, respectively over chemical bleaching. Strategy II was proved to be 35.71% more efficient in ClO₂ saving than conventional method. Significant improvement in various pulp properties viz. tensile strength 25.70%, breaking length 21.80%, burst factor 20.00%, burstness 13.86%, tear factor 6.61% and tearness 18.88%, was also observed through ‘mixed lay out’ strategy.     

Xylanases from <Emphasis Type="Italic">Aspergillus niger,Aspergillus niveus</Emphasis> and <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis> produced under solid-state fermentation and their application in cellulose pulp bleaching

This study describes the production of xylanases from Aspergillus niveus, A. niger, and A. ochraceus under solid-state fermentation using agro-industrial residues as substrates. Enzyme production was improved using a mixture of wheat bran and yeast extract or peptone. When a mixture of corncob and wheat bran was used, xylanase production from A. niger and A. ochraceus increased by 18%. All cultures were incubated at 30 °C at 70–80% relative humidity for 96 h. For biobleaching assays, 10 or 35 U of xylanase/g dry cellulose pulp were incubated at pH 5.5 for 1 or 2 h, at 55 °C. The delignification efficiency was 20%, the brightness (percentage of ISO) increased two to three points and the viscosity was maintained confirming the absence of cellulolytic activity. These results indicated that the use of xylanases could help to reduce the amount of chlorine compounds used in cellulose pulp treatment.     

Optimization of biobleaching of paper pulp in an expanded bed bioreactor with immobilized alkali stable xylanase by using response surface methodology

Purified alkali stable xylanase from Aspergillus fischeri was immobilized on polystyrene beads using diazotization method. An expanded bed bioreactor was developed with these immobilized beads to biobleach the paper pulp in continuous mode. Response surface methodology was applied to optimize the biobleaching conditions. Temperature (degrees C), flow rate of pulp (ml/min) and concentration of the pulp (%) were selected as variables in this study. Optimal conditions for biobleaching process were reaction temperature 60 degrees C, flow rate of 2 ml/min and 5% (w/v) of pulp. The kappa number reduced from 66 in the unbleached pulp to 20 (reduction of 87%). This system proves to be a better option for the conventional chlorine based pulp bleaching.     

TCF mill trial on softwood pulp with Korsäs thermostable and alkaline stable xylanase T6

Abstract: Use of hemicellulases, including xylanases, for delignification in the paper industry has been slowed down by the lack of large-scale availability of enzymes which are active at a high pH (above 8) and a high temperature (above 60°C), conditions prevailing in many bleaching processes. During the past years, acidic or neutral hemicellulases, working at temperatures below 60°C, were used in most mill experiments. The Korsäs T6 xylanase from Bacillus stearothermophilus , which is active at a pH above 9.0 and at a temperature above 65°C, was produced on a large scale in collaboration with Gist-brocades and was employed on a full scale mill trial to produce a Total Chlorine chemical-Free (TCF) pulp from softwood. The bleaching sequence used was (OO)BQQPP. where O stands for oxygen delignification. B for the enzymatic treatment, Q for the chelating agent step and P for the hydrogen peroxide step. The enzyme bleaching step was performed during a period of 4 h at 63 ± 1°C and pH 8.7 ± 0.1. The results of the mill trial show that the TCF pulp produced had a brightness of 78% ISO and, at the same time, it preserved the same strength properties as chlorine dioxide-bleached pulp. The saving of hydrogen peroxide was 20%. The results on brightness, strength and chemical saving of this first full scale trial with T6 xylanase indicate that, after optimization, a TCF bleaching sequence including an enzymatic step with a xylanase working at a high pH and a high temperature, such as T6 xylanase, can be used to produce a high-strength bleached pulp. The advantages of a high pH and a high temperature enzymatic bleaching step are discussed.     

Prebleaching of kraft pulp with full-length and truncated forms of a thermostable modular xylanase from Rhodothermus marinus

Full-length and truncated forms of a modular thermostable xylanase (EC 3.2.1.8., glycoside hydrolase family 10) were used in bleaching sequences of hardwood and softwood kraft pulps. Enzymatic treatment led to brightness gains of all pulps but the result depended on the pulp source. The presence of the additional domains in the full-length enzyme (including carbohydrate-binding modules) did not improve the bleaching process. No significant change in viscosity was seen after enzyme treatments indicating an unaffected pulp fibre length.     

Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3

Streptomyces sp. QG-11-3, which produces a cellulase-free thermostable xylanase (96 IU ml⁻¹) and a pectinase (46 IU ml⁻¹), was isolated on Horikoshi medium supplemented with 1% w/v wheat bran. Carbon sources that favored xylanase production were rice bran (82 IU ml⁻¹) and birch-wood xylan (81 IU ml⁻¹); pectinase production was also stimulated by pectin and cotton seed cake (34 IU ml⁻¹ each). The partially purified xylanase and pectinase were optimally active at 60°C. Both enzymes were 100% stable at 50°C for more than 24 h. The half-lives of xylanase and pectinase at 70, 75 and 80°C were 90, 75 and 9 min, and 90, 53 and 7 min, respectively. The optimum pH values for xylanase and pectinase were 8.6 and 3.0, respectively, at 60°C. Xylanase and pectinase were stable over a broad pH range between 5.4 and 9.4 and 2.0 to 9.0, respectively, retaining more than 85% of their activity. Ca²⁺ stimulated the activity of both enzymes up to 7%, whereas Cd²⁺, Co²⁺, Cr³⁺, iodoacetic acid and iodoacetamide inhibited xylanase up to 35% and pectinase up to 63%; at 1 mM, Hg²⁺ inhibited both enzymes completely. Journal of Industrial Microbiology & Biotechnology (2000) 24, 396–402. Received 29 September 1999/ Accepted in revised form 02 February 2000     

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-E_o-D bleaching sequence.     

Using both xylanase and laccase enzymes for pulp bleaching

Two enzyme treatments involving xylanase (X) and laccase (L) were used jointly in an XLE sequence (where E denotes alkaline extraction) to bleach oxygen-delignified eucalyptus kraft pulp in the presence of 1-hydroxybenzotriazol (HBT) as mediator. The results of the XLE sequence were compared with those of an LE sequence. The application conditions for the laccase–mediator system were optimized by using a sequential statistical plan involving three variables (viz., the laccase and mediator doses, and the reaction time) with both sequences. The models used to predict the kappa number and brightness revealed that, once all accessible lignin was removed, the system altered other coloured compounds. The best conditions for the L stage involved a reduced mediator dose (0.5% odp). The xylanase pretreatment increased the accessibility of residual lignin and facilitated removal of hexenuronic acids. For a specific target brightness level of 70% ISO, the X pretreatment can save as much as 30% laccase and 80% mediator while shortening the reaction time by 45%.     

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.     

Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi

Manganese dependent peroxidase (MnP) is the main enzyme implicated in the biobleaching of kraft pulps by white rot fungi. The goal of this study was to evaluate the Mn requirement for biobleaching of eucalyptus oxygen delignified kraft pulp (OKP) by various white rot fungi: Trametes versicolor, Phanerochaete sordida, Phlebia radiata, Stereum hirsutum and Bjerkandera sp. strain BOS55. All of the strains tested produced MnP and provided extensive bleaching of OKP when 33 μM Mn was included in the medium. Bjerkandera sp. strain BOS55 was the only strain that also displayed MnP production and biobleaching activity of EDTA-extracted OKP in the complete absence of Mn. However, MnP and biobleaching activity in the absence of Mn was dependent on the presence of organic acids in the medium. The fact the biobleaching was correlated to MnP activity irrespective of whether Mn was present or absent suggests that there may be roles for MnP in Bjerkandera under Mn-deficient conditions. Although manganese-independent peroxidase (MIP) and lignin peroxidase (LiP) were also detected, the titres were much smaller in comparison with those of MnP, so their relative role in biobleaching can be predicted to have a minor importance in comparison with MnP. Only in the case of Bjerkandera, was the expression of LiP stimulated in the presence of oxalate but final brightness was not substantially affected.