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Xylanase production from B. megaterium was enhanced using solid state fermentation with respect to the use of solid substrate, moistening solution, moisture content, inoculum, sugars, soyabean meal, amino acids, and extraction with surfactant. An increase of approximately 423-fold in xylanase production and complete suppression of CMCase production was achieved over submerged liquid fermentation. Biobleaching using this cellulase-free xylanase, 8 U/g of oven dried pulp of 10% consistency, showed 8.12% and 1.16% increase in brightness and viscosity, 13.67% decrease in kappa number, and 31% decrease in chlorine consumption at the CD stage.

The efficiency of xylanase of Bacillus brevis BISR-062 as a prebleaching agent was evaluated on three nonwoody pulps at two different pH values (7.0 and 8.5). Crude xylanase was found to have an optimum temperature and pH of 65-70 degrees C and 7.0, respectively. The stability of the enzyme was determined at two pH values (7.0 and 8.0), and it lost approx 50% of its activity at both values within 2 h at 50 degrees C. However, the enzyme was found to be effective as a prebleaching agent only with rice straw pulp. A maximum brightness gain of 6 points was obtained with this pulp at pH 7.0. The strength properties of the rice straw pulp at pH 7.0 also improved as the result of enzyme treatment.

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 degrees 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 degrees C, with a second peak of activity at 90 degrees C. When assayed at pH 8.5, optimum activity was seen at 80 degrees C. The crude enzyme was thermostable at 55 degrees 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 beta-xylosidase and alpha- 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.

The effluent from pulp bleaching processes containing chlorinated lignin and degraded polyphenolic intermediates remains as a major source of water pollution from the pulp and paper industries. Alternative elemental chlorine free bleaching methods based on the usage of chlorine dioxide, ozone and hydrogen peroxide are potential substitutes. Bio-bleaching methods, which involve pre-treatment of pulp with microbial enzymes such as xylanases, have emerged as viable options. Investigations reported in this paper aim at exploring the suitability of commercial bacterial xylanase enzyme preparations for bio-bleaching of bagasse pulps in conjunction with specific chemical bleach sequences employing hydrogen peroxide (P), alkali extraction (E), ozonation (Z), hypochlorite (H) and chelation (Q) stages. The effluent profiles and pulp qualities obtained for each of the bleach combinations (involving bio-bleaching and chemical bleaching sequences) were monitored. Analysis of the results clearly indicates that the inclusion of enzyme pre-treatment with the TCF (total chlorine free) and ECF (elemental chlorine free) sequences has a significant effect on the effluent (COD, lignin and colour) and pulp quality (kappa number, brightness) parameters. In conclusion, the findings of this investigation indicate the potential promise of enzyme pre-treatment in combination with chemical bleaching to enhance the quality of pulps and combined effluents.

The ability to degrade organic solid wastes by the fungus Saccobolus saccoboloides was studied. The organism, unusual in such studies, was cultivated in synthetic liquid media with agitation, and on day 8 of growth the mycelium was passed to flasks with trimming. On day 16 of growth, the trimming degradation was assesed by carboxymethylcellulase, xylanase, and amylase activities evaluation, and NaOH 1% hydrolysis. Later on, the type of waste was modified (trimming, filter paper, newspaper, cardboard, sawdust and wood shaving were used) as well as the mass (300-1800 mg/flask). In these cases the enzymatic activities increased between 300 and 600 mg/flask. The total separation of the cellular components in all types of paper and cardboard was observed, together with a high loss of weight. S. saccoboloides was not able to degrade the wood wastes

To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25.The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose.The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced.The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.

Two enzymatic extracts obtained from xylan-grown Aspergillus terreus CCMI 498 and cellulose-grown Trichoderma viride CCMI 84 were characterised for different glycanase activities. Both strains produce extracellular endoxylanase and endoglucanase enzymes. The enzymes optimal activity was found in the temperature range of 45-60 ºC. Endoglucanase systems show identical activity profiles towards temperature, regardless of the strain and inducing substrate. Conversely, the endoxylanases produced by both strains showed maximal activity at different pH values (from 4.5 to 5.5), being the more acidic xylanase produced by T. viride grown on cellulose. The endoglucanase activities have an optimum pH at 4.5-5.0. The endoxylanase and endoglucanase activities exhibited high stability at 50 ºC and pH 5.0. Mannanase, β-xylosidase, and amylase activities were also found, being the first two activities only present for T. viride extract. These two enzymatic extracts were used for mixed office wastepaper (MOW) deinking. When the enzymatic extract from T. viride was used, a further increase of 24% in ink removal was obtained by comparison with the control. Both enzymes contributed to the improvement of the paper strength properties and the obtained results clearly indicate that the effective use of enzymes for deinking can also contribute to the pulp and paper properties improvement., PRAXIS/BIO/1133/95. BIC/3087/96, BD/3253/96.

The influence of lignin, lignin model compounds, and black liquor from the kraft pulping process on the hydrolysis of xylan by xylanase was investigated. Addition of vanillic acid, acetovanillone, and protocatechuic acid increased the rate of hydrolysis of xylan by as much as 18-50% at low concentrations, but reached maxima at about 0.05% concentration. Addition of vanillin caused a 15% improvement in xylan hydrolysis, while addition of guaiacol more than doubled the hydrolysis rate. Increasing concentrations of either lignin or black liquor also increased the hydrolysis rate of xylan. Circular dichroism spectroscopy indicated a change in the structure of xylanase in the presence of black liquor.

The aim of this work was to extract and to purify xylanase, produced by Bacillus pumilus from the crude fermentation broth, using aqueous two-phase systems (ATPS). The xylanase was extracted by partitioning in ATPS composed of phosphate and polyethylene glycol (PEG). The effect of tie-line length, PEG molecular mass and NaCl concentrations upon the purification factors and yields of xylanase were investigated by statistical design. The best system studied was that containing 22% PEG6000, 10% K2HPO4 and 12% NaCl with a purification factor of 33 and a 98% yield of enzyme activity. This system was also used for continuous extraction in a pulsed caps column. Subsequently, the xylanase from the crude fermentation broth was tested in hardwood kraft pulp bleaching.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA. gave the most effective kappa number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.

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).

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.

Hemicellulase-aided bleaching is the first full-scale biotechnical application in the pulp and paper industry which truly exploits the unique specificity and safety of biocatalysts. Hemicellulases are used to modify the structure of xylan and glucomannan in pulp fibers in order to enhance the chemical delignification. This technology can be combined with various types of kraft pulping processes and bleaching sequences. The aims of the enzymatic treatment depend on the actual mill conditions, and may be related to environmental demands, reduction of chemical costs, or maintenance or even improvement of product quality. The technology is applied on the mill scale in several countries. This review describes the principles of the enzyme-aided bleaching, the composition of the fiber substrates, the basic enzymology involved, and the present knowledge of the mechanisms of the action of enzymes, as well as the practical results and advantages obtained on the laboratory and industrial scale.

Xylan is the main carbohydrate found in the hemicellulosic fraction of plant tissues and accounts for one third of all renewable organic carbon available on earth. Xylanase, the major component of an enzymatic consortium, acts in nature by depolymerizing xylan molecules into monomeric pentosan units that are used by bacterial and fungal populations as a primary carbon source. Xylanase producers have been isolated from all ecological niches where plant material is deposited, and microorganisms often contain multiple loci encoding overlapping xylanolytic functions. The numerical excess of genes and the extensive sharing of structural features within beta-glycanase families suggests that extensive gene duplication and conversion events have occurred during xylanase evolution. Hydrolysis of beta-glycosidic linkages is sponsored by a general acid catalytic reaction common to all glycanases, whereas substrate recognition is specified by subsites that interact with adjacent glycosyl units. Under natural conditions xylanases are inducible by the products of their own action and subject to carbon catabolite repression. Bleaching paper pulps with xylanases is the first successful commercial application for these enzymes. The recovery of cellulosic textile fibers is the next logical application and bioconversion of biomass into fuels and chemicals, remains the ultimate target. Recent developments have shown that metabolic pathways can be transferred from one organism to another and proteins can be modified to gain conformational stability, suggesting that naturally occurring systems can be custom engineered to the situation in the fermentation tank. Thus, biotechnologies developed to transform biomass into marketable products that gradually substitute materials derived from non-renewable resources are becoming commercially worthwhile.