Cellulase-free xylanases from Bacillus and other microorganisms

Xylanases are used mainly in the pulp and paper industries for the pretreatment of Kraft pulp prior to bleaching to minimize use of chlorine, the conventional bleaching agent. This application has great potential as an environmentally safe method. Hydrolysis by xylanases of relocated and reprecipitated xylan on the surface of cellulose fibres formed during Kraft cooking facilitates the removal of lignin by increasing permeability to oxidising agents. Most of the xylanases reported in the literature contained significant cellulolytic activity, which make them less suitable for pulp and paper industries. The need for large quantities of xylanases which would be stable at higher temperatures and pH values and free of cellulase activity has necessitated a search for novel enzymes. We have isolated and characterised several xylanase-producing cultures, one of which (an alkalophilic Bacillus SSP-34) produced more than 100 IU ml(-1) of xylanase activity. The SSP-34 xylanases have optimum activity at 50 degrees C in a pH range 6-8, with only small amounts of cellulolytic activity (CMCase (0.4 IU ml(-1), pH 7), FPase (0.2 IU ml(-1), pH 7) and no activity at pH 9).     

Production of a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> QM9414 xylanase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its application in biobleaching of wheat straw pulp

The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was 5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile, burst and tear strength of pulp fibers.     

Application of crude xylanase from Bacillus licheniformis 77-2 to the bleaching of eucalyptus Kraft pulp

Alkalophilic Bacillus licheniformis 77-2 produced an extracellular alkali-tolerant xylanase with negligible cellulase activity in medium containing corn straw. The effectiveness of crude xylanase on treatment of eucalyptus Kraft pulp was evaluated. A biobleaching experiment was carried out to compare the chlorine saving with pulp treated and untreated by the enzyme. Two-stage bleaching was employed, using a ClO₂ chlorination and NaOH extraction (DE sequence). With the enzymatic treatment, in order to obtain the same value of Kappa number and brightness, respectively 28.5 and 30% less ClO₂ was required in comparison to the enzymatically untreated samples.     

Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l(-1)), improved FAEA activity 24.5-fold and a yield of 1 g l(-1) of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification--close to 75%--and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector.     

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.     

重组极耐热木聚糖酶和重组极耐热漆酶协同漂白麦草浆

利用来自海栖热袍茵的重组极耐热木聚糖酶XynB和来自嗜热栖热菌Thermus thermophilus HB27的重组极耐热漆酶Tth-laccase对麦草浆进行协同漂白。结果表明,当未漂浆经XL漂序处理(X:重组木聚糖酶用量20 U/g绝干浆,pH 5.8,温度90℃,浆浓8%,处理时间2 h;L:重组漆酶用量3 U/g绝干浆,pH 4.5,温度90℃,浆浓8%,处理时间1.5 h),可获得最佳漂白效果。与对照浆比较,XL处理使浆料白度提升11.5%ISO,卡伯值降低6.9。双酶协同处理在改善浆料可漂性的同时,对纸浆纤维强度无负面影响。在后续过氧化氢漂白段中,当漂终白度相近时,XL预处理浆可节省约50%H_2O_2消耗量。     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Production of cellulase‐free xylanase by the recombinant Bacillus subtilis and its applicability in paper pulp bleaching

A metagenomic xylanase gene (Mxyl) was successfully cloned into shuttle vector pWH1520 and expressed in Bacillus subtilis extracellularly. On induction with xylose, recombinant xylanase secretion commenced after 6 h. Identifying critical variables for recombinant xylanase production by one‐variable‐at‐time approach followed by optimization of the selected variables (xylose, inoculum density, incubation density) by response surface methodology (RSM) led to three‐fold enhancement in extracellular xylanase production (119 U mL^?1). When the pulp was treated with recombinant xylanase at 80°C and pH 9.0, kappa number of the pulp was reduced with concomitant increase in brightness and 24% reduction in chlorine consumption. This is the first report on the expression of metagenomic xylanase gene in Bacillus subtilis extracellularly and its utility in developing an environment‐friendly pulp bleaching process. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1441–1447, 2013     

Production,partial characterization and use of fungal cellulase-free xylanases in pulp bleaching

Seven fungal strains were screened for their ability to produce cellulase-free xylanases that could be used in pretreatment of sulphite pulp prior to bleaching. The potential xylanase producers were subjected to shake flask fermentations using four different carbon sources: wheat bran, corn cobs, oat spelts xylan and bleach plant effluent. When grown on corn cobs, Aspergillus foetidus (ATCC 14916) produced significant levels of xylanase (547.4 U/ml), accompanied however by 6.6 U/ml of cellulase activity. Two other strains, Aspergillus oryzae (NRRL 1808) and Gliocladium viride (CBS 658.70), produced high yields of cellulase-free xylanase on oat spelts xylan. The crude enzymes of these two isolates were characterized with respect to pH and temperature optima and stability in order to standardize the optimum conditions for their use on pulp. Although the two xylanases differed in their abilities to remove reducing sugars from pulp, their biobleaching abilities, when assessed in hydrogen peroxide delignification of pulp, were very similar: both of them increased brightness by 1.4 points and removed 7% of hemicellulose from pulp.     

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.     

Optimization of xylanase production from Aspergillus niger for biobleaching of eucalyptus pulp

A crude endo-xylanase produced by Aspergillus niger BCC14405 was investigated for its potential in pre-bleaching of chemical pulp from eucalyptus. The optimal fermentation conditions on the basis of optimization using response surface methodology included cultivation in a complex medium comprising wheat bran, rice bran, and soybean meal supplemented with yeast extract, glucose, peptone, and lactose with a starting pH of 6.0 for 7 d. This resulted in production of 89.5 IU/mL of xylanase with minor cellulase activity. Proteomic analysis using LC/MS/MS revealed that the crude enzyme was a composite of hemicellulolytic enzymes, including endo-β-1,4-xylanase and other hemicellulolytic enzymes attacking arabinoxylan and mannan. Pretreatment of the pulp at a xylanase dosage of 10 IU/g increased the brightness ceiling after the C-Eop-H bleaching step up to 3.0% using a chlorine charge with a C-factor of 0.16-0.20. Xylanase treatment also led to reduction in chlorine charge of at least 20%, with an acceptable brightness level. The enzyme pretreatment resulted in a slight increase in pulp viscosity, suggesting an increase in relative cellulose content. The crude enzyme was potent in the enzyme-aided bleaching of chemical pulp in an environmentally friendly pulping process.     

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.     

Production and partial characterization of cellulase free xylanase by Bacillus subtilis C 01 using agriresidues and its application in biobleaching of nonwoody plant pulps

AIMS: To optimize the solid-state cultivation conditions for xylanase production using agriresidues and testing the biobleaching efficiency of xylanase on nonwoody plant fibre materials. METHODS AND RESULTS: An extracellular cellulase free xylanase was produced from Bacillus subtilis C 01 using various inexpensive substrates under solid-state cultivation. High level of xylanase production (135 IU gds(-1)) was observed when grown on wheat bran followed by maize powder (50 IU gds(-1)). The maximum xylanase (136 IU gds(-1)) production was occurred in wheat bran-to-moisture ratio of 1 : 1 at 72 h. The xylanase pretreated pulp samples of banana, silk cotton and cotton showed an increased brightness of 19.6, 11.6 and 7.9%, respectively. CONCLUSIONS: The enzyme-aided biobleaching results indicate that the xylanase has potential application in enhancing the brightness of nonwoody plant fibre pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on biobleaching of banana fibres, silk cotton and cotton pulps using xylanase. The biobleaching results of secondary fibres are promising and can be transferred to paper mills, which utilize nonwoody plant fibres as a raw material for paper production.     

Characterization of a thermostable and alkaline xylanase from Bacillus sp. and its bleaching impact on wheat straw pulp

Delignification efficacy of xylanases to facilitate the consequent chemical bleaching of Kraft pulps has been studied widely. In this work, an alkaline and thermally stable cellulase-less xylanase, derived from a xylanolytic Bacillus subtilis, has been purified by a combination of gel filtration and Q-Sepharose chromatography to its homogeneity. Molecular weight of the purified xylanase was 61 kDa by SDS–PAGE. The purified enzyme revealed an optimum assay temperature and pH of 60°C and 8.0, respectively. Xylanase was active in the pH range of 6.0–9.0 and stable up to 70°C. Divalent ions like Ca²⁺, Mg²⁺ and Zn²⁺ enhanced xylanase activity, whereas Hg²⁺, Fe²⁺, and Cu²⁺ were inhibitory to xylanase at 2 mM concentration. It showed K _m and V _max values of 9.5 mg/ml and 53.6 μmol/ml/min, respectively, using birchwood xylan as a substrate. Xylanase exhibited higher values of turn over number (K _cat) and catalytic efficiency (K _cat/K _m) with birchwood xylan than oat spelt xylan. Bleach-boosting enzyme activity at 30 U/g dry pulp displayed the optimum bio-delignification of Kraft pulp resulting in 26.5% reduction in kappa number and 18.5% ISO induction in brightness at 55°C after 3 h treatment. The same treatment improved the pulp properties including tensile strength and burst index, demonstrating its potential application in pre-bleaching of Kraft pulp.     

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.     

Changes in the structural composition of hardwood kraft pulp during bleaching

The possibility of using xylanase preparations for hydrolyzing hemicelluloses in a non-bleached kraft pulp in order to facilitate its bleaching was studied. The effects of enzymatic preparations of fungal and bacterial origins were examined, and the optimal conditions for xylanase activity were determined. UV spectroscopy demonstrated that the treatment of kraft pulp with enzymatic preparations containing xylanase facilitated the subsequent removal of lignin and increased the brightness by 5%. The effect of enzymatic treatment was retained in the case of peroxide bleaching. The enzymatic preparations studied are promising for the development of chlorine-free pulp bleaching technologies.     

Natural and recombinant fungal laccases for paper pulp bleaching

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l^–1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K _m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Construction of a xylan-fermenting yeast strain through codisplay of xylanolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells

Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on alpha-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and beta-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.