Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.     

Trichoderma Xylanases,Their Properties and Application

Abstract

Trichoderma spp. are known to produce enzymes with high xylanolytic activity. Different xylanases and various components of their xylanolytic system have been identified and purified. Some of the xylanases have been characterized extensively with respect to their physicochemical, hydrolytic, and molecular properties. Cellulase-free xylanase preparations have been tested successfully in industrial applications such as the prebleaching of kraft pulps in the pulp and paper industry. Future work on understanding the functional significance of xylanase multiplicity, the mechanisms of xylanase prebleaching, and the structural conformation of xylanases could lead to improved or alternative applications of Trichoderma xylanases.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp

A thermoalkalophilic and cellulase-free xylanase produced from Arthrobacter sp. MTCC 5214 by solid-state fermentation using wheat bran as a carbon source was evaluated for prebleaching of kraft pulp. The UV absorption spectrum of the compounds released by enzyme treatment showed a characteristic peak at 280 nm, indicating the presence of lignin in the released colouring matter. Enzymatic prebleaching of kraft pulp showed 20% reduction in kappa number of the pulp without much change in viscosity. Enzymatic treatment reduced the amount of chlorine by 29% without any decrease in brightness. The viscosity of xylanase treated pulp was 4.0 p, whereas the viscosity of the pulp treated exclusively with chlorine was 4.1 p.     

Recombinant expression, characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Effects of dietary cellulase and xylanase addition on digestion,rumen fermentation and methane emission in growing goats

The objective of this study was to evaluate the effectiveness of supplementation of cellulase and xylanase to diets of growing goats to improve nutrient digestibility, utilisation of energy and mitigation of enteric methane emissions. The experiment was conducted in a 5 × 5 Latin square design using five goats with permanent rumen fistulae and five treatments consisted of two levels of cellulase crossed over with two levels of xylanase plus unsupplemented Control. The cellulase (243 U/g) derived from Neocallimastix patriciarum was added at 0.8 and 1.6 g/kg dry matter intake (DMI) and the xylanase (31,457 U/ml) derived from Aspergillus oryzae was fed at 1.4 and 2.2 ml/kg DMI. There were no differences in apparent digestibility of organic matter, neutral detergent fibre, acid detergent fibre and rumen fermentation parameters (i.e. ammonia-nitrogen [N], volatile fatty acids) among all treatments. Dietary cellulase and xylanase addition did not influence energy and N utilisation. But compared to xylanase addition at the higher dose, at the low xylanase dose the retained N, the availability of retained N and digested N were increased (p < 0.01). Moreover, enzyme addition did not affect the enteric methane emission and community diversity of ruminal methanogens. The present results indicated that previous in vitro findings were not confirmed in ruminant trials.     

Cloning and Sequence Analysis of Three Variants of the Gene Encoding Alkaline Xylanase C from the Alkaliphilic Bacillus sp. (NCL 87-6-10)

Alkaline xylanase C from the alkaliphilic Bacillus sp. (NCL 87-6-10) has a low molecular weight and alkaline pI and is cellulase-free, properties compatible with its use in the prebleaching of pulp. We report here the cloning and sequence analysis of three variants of the gene encoding xylanase C; xyl C1, xyl C2, and xyl C3. In phylogenetic analysis, the three xylanase C variants clustered into a single group along with other reported alkaline xylanases. Residues contributing to the alkaline pH were present in all three variants. DNA and protein sequence comparison of these variants with other reported alkaline xylanases revealed silent mutations, some of which are due to codon preference in the respective organisms. The recombinant Xyl C1 that was successfully expressed in E. coli BL21 (DE3) had properties similar to the native enzyme.     

Studies on cell wall loosening enzymes in hormone treated internodes of <Emphasis Type="Italic">Merremia emarginata</Emphasis>

The cell wall loosening enzymes viz. glycosidases, polygalacturonase and xylanase were analyzed in cytoplasmic and wall bound fractions extracted from control and hormone (GA₃ NAA, PAA) treated internodes, as they are known to play a key role in cell wall metabolism. Among the glycosidases, wall bound β-glucosidase and α-galactosidase activities were significantly correlated with age of control internodes. Cytoplasmic α-galactosidase showed significant correlation in hormone treated internodes. Maximum correlation was observed in GA₃, followed by PAA and NAA. Wall bound xylanase activity was well correlated with length only in NAA treated internodes and less after GA₃ treatment while cytoplasmic xylanase showed correlation with intrnode length only in control and after NAA treatment. Cytoplasmic polygalacturonase showed correlation with internode length only after GA₃ treatment while wall bound polygalacturonase showed correlation with internode length after NAA treatment. The possible role of these enzymes in internode development is discussed.     

Cellulase-free xylanase production from an alkalophilicBacillus species

An alkalophilicBacillus (NCL-87-6-10, NCIM 2128), with a high productivity for extracellular xylanase (EC 3.2.1.8) and free of cellulase, was isolated from soil containing coconut fibre detritus. When grown on a wheat bran/yeast extract medium in submerged culture for 48 h, it produced 100 to 120 IU of enzyme activity per ml. The crude enzyme consists of two fractions of apparent mol sizes of approx 10.4 and 29 kDa in the proportion of 90:10, as determined by native gel exclusion chromatography. Optimum activity of the xylanase was at 60°C and pH 8.0. A two-fold increase in enzyme activity was obtained when reducing agents, thioethanol and dithiothreitol, were included in the assay.NCL Communication No. 5381.     

Identification of a Major Xylanase from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> as a 14-kD Protein

Aspergillus flavus K49 secreted at least two xylanase activities when grown on a medium containing larch (wood) xylan as a sole carbon source. Enzyme activity was assayed using an agar medium containing Remazol Brilliant Blue R conjugated oat spelt xylan as substrate. Crude enzyme preparations were inhibited by Hg⁺², with an ED₅₀ of 17.5 mM and maximum inhibition of 83% at 50 mM. A concentrated sample of A. flavus K49 xylanase preparation was subjected to gel filtration chromatography on a P-30 column. A small protein peak coinciding with the major peak of xylanase activity was separated from the other secreted fungal proteins. An additional peak of xylanase activity was observed in fractions containing multiple fungal proteins. Analysis by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of fractions containing the smaller molecular weight xylanase revealed a major and minor protein band in the vicinity of 14 kD. Analysis of these same fractions by acidic native PAGE revealed a single band. Confirmation of identity for the isolated xylanase was provided by isolation of a protein band from a SDS–PAGE gel, followed by trypsin digestion/analysis by tandem mass spectrometry. Comparison of the peptide library derived from this protein band with sequence data from the A. oryzae genomic data base provided a solid match with an endo-1,4-β-xylanase, XlnA. This identification is consistent with a low molecular weight protein associated with the major xylanolytic activity. XlnA may be a highly mobile (diffusible), plant wall hemicellulose degrading factor with significant activity during plant infection.     

The effects of xylanase type enzymes on the different layers of birch wood ORGANOSOLV pulp fibre walls

Some effects of the xylanase treatment on the separate birch ORGANOSOLV pulp fibre wall morphological layers were examined. These investigations were focused on the outer layers, i.e. the primary wall (P) and the outer layer of the secondary wall (S₁), as well as the central layers, i.e. the central layer of the secondary wall (S₂) and the tertiary wall (T). Step by step, the fractionation of the pulp components in the polar solvents N,N-dimethylformamide (DMFA), dimethylsulphoxide (DMSO) and DMSO/H₃PO₄ was used as a mild technique for the isolation of the lignin-carbohydrate complexes. The different residual amounts of lignin and hemicelluloses in the outer and central pulp fibre wall layers as well as the different lignin-hemicellulose ratios were determined. The size-exclusion chromatographical (SEC) analysis showed a higher initial lignin content in the region of the high molecular mass (MM) fibre wall fraction extracted with “DMSO/H₃PO₄” than the outer cell wall layers. In the central layers, the amounts of soluble lignin (calculated on the mass of total dissolved substance) were approximately the same for all the three solvents. The xylanase treatment brought the most considerable changes in the high MM part of the residual lignin (the lignin carbohydrate complex). This was true for both the P-S₁ and S₂-T layers. The careful brightness comparison of the outer and central fractions after the X-E-P-P bleaching sequence showed a surprisingly low bleachability of the outer layer fraction. The xylanase action depended on the composition of the lignin-carbohydrate complex (LCC) and the extent of the maintenance of the outer layers during the pulping process.     

The effects of crop rotation and fertilization on soil xylanase activity in a soil of the southeastern United States

Summary Xylanase activity was determined in soils from a 3-year rotation with corn, (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean (Glycine max (L) Merr.) as summer crops at Auburn, Alabama, in the southeastern U.S.A. Winter wheat (Triticum aestivum L.) followed corn and a period of winter fallow as maintained after soybeans. A combination of common vetch (vicia sativa L.) and crimson clover (Trifolium incarnatum Gibelli & Belli) followed cotton during the winter months to serve as green manure. Highest xylanase activity was detected after soybeans and lowest after cotton among the summer crops. The culture of wheat and winter legumes resulted in increased soil xylanase activity wheareas winter fallowing reduced the activity. The effect of various fertilization schemes superimeposed on the rotation on soil xylanase activity was also studied. Seasonal fluctuations in soil xylanase activity were not affected by the fertilization regimes and highest xylanase activities were observed during crop periods and with fertilization treatments that resulted in high root densities but not necessarily in high yields.     

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.     

Purification and characterization of a cellulase-free xylanase of a moderate thermophile Bacillus licheniformis A99

Two cellulase-free xylanases were secreted by a thermophile, Bacillus licheniformis A99. Of the two, the predominant one was purified to homogeneity. The enzyme was optimally active at 60 °C, pH 6–7.5, and had a molecular weight of about 45 KDa and isoelectric point of 7.0 ± 0.2. The K _m (for birchwood xylan) and V _max were 3.33 mg/ml and 1.111 mmols mg^–1 protein min^–1 respectively. The half-life of the enzyme was 5 h at 60 °C. All cations except Hg²⁺ and Ag⁺ as well as EDTA were well tolerated and did not adversely affect xylanase activity. However, SDS inhibited the enzyme activity. The release of reducing sugars from unbleached commercial pulp sample on treatment with the enzyme indicated its potential in prebleaching of paper pulp. The enzyme caused saccharification of lignocellulosics such as wheat bran, wheat straw and sawdust. This is the first report on purification and characterization of cellulase-free xylanase from a moderate thermophile Bacillus licheniformis.     

Enzymatic prebleaching of sulphite pulps

The ability of a crude enzyme preparation ofAureobasidium pullulans containing xylanase [61 units (U)/ml] and xylosidase (3 U/ml) activity to remove pentosans from unbleached sulphite pulps was investigated. Greater amounts of pentosans and reducing sugars were released from the pulp when the enzyme dosages and incubation times were increased. A combination of enzyme hydrolysis and alkali extraction resulted in a greater removal of pentosans than using the enzyme preparation alone. By treatment with a xylanase loading of 450 U/g pulp for 24 h followed by alkaline extraction, 35% of the pentosans were removed. The kappa number decreased up to 30% whereas viscosity was only slightly affected by these treatments. Enzymatic hydrolysis released mainly xylose whereas xylobiose was the main product liberated by alkaline extraction. Scanning electron micrographs indicated improved fibrillation and flexibility of the fibre structure by enzyme treatment.     

Recombinant expression,characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Transformation and expression of an anaerobic fungal xylanase in several strains of the rumen bacterium Butyrivibrio fibrisolvens

AIMS: To obtain reliable transformation of a range of Butyrivibrio fibrisolvens strains and to express a Neocallimastix patriciarum xylanase gene in the recipients. METHODS AND RESULTS: Eight strains (H17c, E14, LP1309, LP1028, AR11a, OB156, LP210B and LP461A) of Bu. fibrisolvens were transformed by the Gram-positive vector pUB110. A xylanase expression/secretion cassette containing Bu. fibrisolvens promoter and signal peptide elements fused to catalytic domain II of the N. patriciarum xylanase A cDNA (xynANp) was inserted into pUB110 to create the plasmid pUBxynA. pUBxynA was used to transform seven of the Bu. fibrisolvens strains transformed by pUB110. In strain H17c pUBxynA, which produced native xylanase, 2.46 U mg-1 total xylanase activity was produced with 45% extracellular xylanase. In strain H17c pUMSX, 0.74 U mg-1 total xylanase activity was produced with 98% extracellular xylanase. H17c pUBxynA exhibited increased (28.7%) degradation of neutral detergent fibre compared with unmodified H17c; however, progressive loss of pUBxynA was observed in long-term cultivation. CONCLUSIONS: A stable transformation system was developed that was applicable for a range of Bu. fibrisolvens strains and high levels of expression of a recombinant xylanase were obtained in H17c which lead to increased fibre digestion. SIGNIFICANCE AND IMPACT OF THE STUDY: This stable transformation system with the accompanying recombinant plasmids will be a useful tool for further investigation aimed at improving ruminal fibre digestion.     

Improvement in the bleaching of kraft pulp with xylanase from Penicillium crustosum FP 11 isolated from the Atlantic forest

Xylanase produced from the newly isolated Penicillium crustosum FP 11 and its potential in the prebleaching of kraft pulp were evaluated using a statistical approach. A Plackett–Burman design (PBD) was carried out to select the significant variables of the medium, these being NaNO₃, KH₂PO₄, MgSO₄, KCl, Fe₂(SO₄)₃, yeast extract, corn stover, and initial pH, in a liquid culture under static conditions for 6 d at 28?°C. Statistical analysis with a central composite design and response surface methodology showed that 0.15% (w/v) KH₂PO₄, 2% (w/v) corn stover, and an initial pH of 6.0 provided the best conditions for xylanase production. Furthermore, xylanase from P. crustosum FP 11 was effective in the bleaching of Eucalyptus kraft pulp, with a significant kappa efficiency of 35.04%. Therefore, the newly isolated P. crustosum FP 11 from the Atlantic Forest biome in Brazil showed two advantages: xylanase production with agricultural residue (corn stover) as a carbon source and an improvement in the bleaching of kraft pulp. Environmental pollution could thus be minimized because of a reduction in the use of chlorine as a bleaching agent.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

Isolation, characterization and fibre degradation potential of anaerobic rumen fungi from cattle

A total of 20 fungal cultures were isolated from the rumen of cattle fed a high fibre-containing diet. All of the isolates showed polycentric growth patterns and were identified as different strains of Orpinomyces and Anaeromyces. Enzyme assays of most of the isolates showed the highest carboxymethylcellulase (CMCase) and xylanase activities after 96 h of growth and highest avicelase activity after 120 h. Among all enzymes tested, xylanase activity was the highest, followed by CMCase and avicelase. The results of the in vitro fibre digestibility and rumen fermentation analyses revealed that the addition of fungal cultures significantly increased acetate, in vitro dry matter digestibility, partition factor values and microbial biomass synthesis levels. Overall, Orpinomyces spp. were found to be the better enzyme producers and fibre degraders than Anaeromyces spp.