Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

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. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Enzymatic accessibility of xylans in lignocellulosic materials

The hydrolysis of fibre-bound and isolated xylans from both birch and pine wood and kraft pulps was studied using purified xylanolytic enzymes of Trichoderma reesei. Despite high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 20% of the theoretical value, apparently due to limited accessibility of the substrates. The fibre-bound xylans were as equally accessible in softwood as in hardwood pulps. The isolated xylans of wood and kraft pulps could be solubilized more extensively, with a hydrolysis yield of 50–65%. The substitution degree of the isolated xylan substrates was reflected in the different hydrolysis yields obtained by the two xylanases, with isoelectric point (pI) values of 9.0 and 5.5. On the more substituted substrates, i.e. pine kraft xylan and pine wood xylan, the two enzymes acted almost similarly, whereas on the less substituted xylan substrates, such as isolated birch kraft xylan, the pI-9.0 enzyme was more efficient. The side-group-cleaving enzymes increased only moderately the solubilization of the substrates.Correspondence to: L. Viikari     

Xylanase production by a new alkali-tolerant isolate of Bacillus

The xylanolytic system of an alkali-tolerant Bacillus sp. consists of several xylanases ranging from 22 to 120 kDa and pI values from 7.0 to 9.0. Crude xylanase retained 72% of initial activity after 5 h at pH 9.0 and 45°C. Xylanase production was induced by xylose and xylan and was maximum at 42°C and pH 7.8. Crude xylanase released xylotriose and xylotetraose as main products of xylan hydrolysis. Xylose was not detected. © Rapid Science Ltd. 1998     

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.     

Xylanases in bleaching: From an idea to the industry

Abstract: The utilization of hemicellulases in bleaching of kraft (sulphate) pulp is considered as one of the most important new large-scale industrial applications of enzymes. This is partly due to the great potential of an environmentally safe method. This method has in a short period also proven to be economically realistic. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-/gb-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable. The hydrolysis of xylan or mannan in the inner fibre layers may also enhance the bleachability. In practical process conditions, properties of the enzymes such as substrate specificity and the pH and temperature optima are of utmost importance. The benefits obtained by enzymes are dependent on the chemical bleaching sequence used as well as on the residual lignin content of pulp. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process and consequently lowering the AOX of the effluents. Enzymes have been applied as a pretreatment both in conventional (C/D)EDED and in ECF (elementary chlorine-free) bleaching sequences. In the production of TCF (totally chlorine-free) pulps, enzymes have also been successfully used for increasing the brightness of pulp.     

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.     

Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivatedon media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulose was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-l) fermentors. Downstream processing of the xylanase-rich, low-cellulose culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-l pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. Correspondence to: M. J. Bailey     

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.     

A cellulase-free xylanase from alkali-tolerantAspergillus fischeri Fxn1

Summary An alkali-tolerant fungusAsperqillus fischeri Fxn1 isolated from xylan enrichment grew in the pH range 5–10 and secreted an extracellular cellulase-free xylanase. Arabinose, lactose, maltose, cellobiose and glucose induced low levels of xylanase (1.8–9.0 IU/ml), whereas xylose, xylan and wheat bran induced higher level (34–45 IU/ml).CMcellulose and FPcellulose did not support growth. The optimum pH of xylanase was 6.0–6.5 and it was stable in a wide range of pH 5–9.5. The optimum temperature was 60°C and it was stable upto 55°C. The half-lives at 50 and 55 °C were 240 and 40 min. respectively. This enzyme released reducing sugars from pulp at pH 9.0 and 40°C.     

Screening of cellulase-free fungal xylanases and evaluation of their performance on sulfite dissolving pulp

Cellulase-free xylanase preparations of different fungal origin lowered the xylan content of acid sulfite dissolving pulp from 5% (w/v) to less than 4%. This was not, though, related to the amount of reducing sugars released. The pulp bleachability improved in samples where xylan had been mostly degraded, however, the increment in brightness declined (from 2.0–2.9 to 1.0–1.2 brightness points) when the subsequent number of chemical bleaching steps increased from 1 to 5.     

Untersuchungen zur Bildung extrazellulärer Hemicellulasen durch Pseudocercosporella herpotrichoides in vitro

Investigations on the production of extracellular hemicellulases by Pseudocercosporella herpotrichoides in vitro For all 15 isolates of Pseudocercosporella herpotrichoides investigated, xylanase as well as arabanase activity could be demonstrated. After cultivation of 3 weeks, the activity of the enzymes reached a peak. The activity of xylanase was considerably increased by addition of xylan in comparison to Maltzin as the sole source of carbohydrate. Also the arabanase activity could be increased significantly by addition of araban or xylan as compared to the Maltzin variant. The optimum temperature with regard to activity and stability of xylanase ranged at 50°C. The pH-optimum for xylanase activity was found to be at pH 5.0, and the enzyme was stable in ° range between pH4.0 and 8.0 (9.0). In case of arabanase, the temperature optimum varied between 40 and 50°C; up to this temperature, the enzyme was also stable. At pH 5.0, the arabanase activity reached its optimum; stability was observed in - pH range between 4.0 and 9.0. In extracts prepared from autoclaved wheat coleoptiles which were inoculated with Pseudocercosporella herpotrichoides, the presence of the enzymes xylanase, arabanase, cellulase and polymethylgalacturonase could be demonstrated. The enzyme activities of the inoculated samples were considerably higher than those of non-inoculated controls. The differences, in most cases, were statistically significant. Der Deutschen Forchungsgemeinschaft danken wir für finanzielle Unterstützung.     

Purification and characterization of a new xylanase (xylanase C) produced by Streptomyces lividans 66

Summary A third extracellular xylanase produced by Streptomyces lividans 66 was isolated from a clone obtained by shotgun cloning through functional complementation of a xylanase- and cellulase-negative mutant using the multicopy vector pIJ702. This enzyme, designated xylanase C, has a relative molecular mass of 22000 and acts on xylan similarly to xylanase B as an endo-type xylanase producing short-chain oligoxylosides. Its specific activity determined at 1100 IU·mg^–1 of protein corresponds on a molecular basis to that of xylanase B and is about three times that of xylanase A. The enzyme shows optimal activity at pH 6.0 and 57°C, values that correspond closely to those observed previously for xylanase A and B. Xylanase C appears not to be glycosylated and has a pI > 10.25. Its K _m and V _max on birchwood xylan are 4.1 mg·ml^–1 and 3.0 mol·min^–1·mg^–1 of enzyme respectively. Whereas specific antibodies raised against xylanase A show no cross-reaction with either xylanase B or with xylanase C, the anti-(xylanase C) antibodies react slightly with xylanase B but not with xylanase A. A comparison of hydrolysis products obtained by reacting individually the three enzymes with birchwood xylan showed characteristic endo-activity patterns for xylanases B and C, whereas xylanase A hydrolysed the substrate preferentially into xylobiose and xylotriose. Sequential xylanase action on the same substrates showed synergistic hydrolysis only when endo-xylanase activity was followed by that of xylanase A.     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

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).     

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.     

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.     

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of -1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylooligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps. Correspondence to: J. N. Saddler     

Purification and properties of xylanase A from alkali-tolerant Bacillus sp. strain BP-23.

Xylanase A from the recently isolated Bacillus sp. strain BP-23 was purified to homogeneity. The enzyme shows a molecular mass of 32 kDa and an isoelectric point of 9.3. Optimum temperature and pH for xylanase activity were 50 degrees C and 5.5 respectively. Xylanase A was completely inhibited by N-bromosuccinimide. The main products of birchwood xylan hydrolysis were xylotetraose and xylobiose. The enzyme was shown to facilitate chemical bleaching of pulp, generating savings of 38% in terms of chlorine dioxide consumption. The amino-terminal sequence of xylanase A has a conserved sequence of five amino acids found in xylanases from family F.