Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp

Fibre-bound and isolated galactoglumanans from pine-wood and pine kraft pulp were hydrolysed with purified mannanases from Trichoderma reesei and Bacillus subtilis. The isolated galactoglucomannans from both wood and pulp could be hydrolysed fairly extensively with both enzymes. In addition to mixed oligomers, the fungal mannase produced mannobiose as the main hydrolysis product whereas the bacterial mannanase produced mannobiose, mannotriose and mannotetraose. Both enzymes hydrolysed the native galactoglucomannan in finely ground pinewood, whereas galactoglucomannan in pine kraft pulp was only hydrolysed by the T. ressei mannanase. Thus, mannanases exhibit different specificities on fibre-bound, modified substrates. In spite of the high enzyme loading, the degree of hydrolysis of fibre-bound substrates did not exceed 10% of the theoretical, probably due to poor accessibility of the substrates. Correspondence to: M. Rättö     

The role of two Trichoderma reesei xylanases in the bleaching of pine kraft pulp

Summary The two major xylanases of Trichoderma reesei with different pI values and pH optima were compared for increasing the bleachability of pine kraft pulp. The efficiencies of the two enzymes acting on pulp substrate were very similar in hydrolysis yield, extraction kappa number or final brightness value. Only slight synergism between the two enzymes was observed in both hydrolysis and bleaching tests. The pH optimum of the pI 5.5 xylanase was similar in pulp treatment and in the hydrolysis of isolated substrates, and the bleaching result also correlated well with the hydrolysis of pulp xylan. By contrast, the pI 9.0 xylanase acted differently on pulp than on isolated xylans at different pH values and the pH optimum on pulp was increased. The bleachability of pulp by the pI 9.0 xylanase was improved more than expected at pH 7.0, although the hydrolysis of pulp xylan was substantially decreased. A similar phenomenon was also observed when the hydrolysis was performed in water instead of buffer. It thus appears that the degree of hydrolysis needed to obtain improved bleachability with pI 9.0 xylanase can be minimized by proper adjustment of the hydrolysis conditions. Correspondence to: J. Buchert     

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     

Investigation of lignin-carbohydrate complexes in kraft pulps by selective enzymatic treatments

The occurrence of covalent bonds between residual lignin and polysaccharides in birch and pine kraft pulps was investigated by specific enzymatic treatments. Pure enzymes degrading cellulose, xylan and mannan were used both separately and in combination. Comparison of the molar masses of polysaccharides and lignin in the orginal pulps and in the residual pulps after enzymatic treatments showed that residual lignin in birch kraft pulp is linked at least to xylan. A minor portion may also be linked to cellulose. In pine kraft pulp some of the residual lignin appears to be linked to cellulose, glucomannan and xylan. The linkages between lignin and cellulose and hemicelluloses may be either native or formed during pulp processing. The results also provided new information on the synergistic action of cellulose- and hemicellulose-degrading enzymes on pulp fibres. The synergism appears to be mainly due to the structure of the pulp fibres, with different layers of cellulose sheets, hemicelluloses and lignin. On the other hand the results also provided information about fibre structure. The degradation of xylan clearly enhanced the action of enzymes on cellulose, suggesting that xylan partially covers the cellulose. A similar phenomenon was not observed in the simultaneous hydrolysis of glucomannan and cellulose. However, the results suggest that glucomannan does interact with cellulose, possibly by non-covalent linkages. Received: 8 July 1998 / Received revision: 7 October 1998 / Accepted: 11 October 1998     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Structure of hardwood glucuronoxylans: modifications and impact on pulp retention during wood kraft pulping

The behaviour of different hardwood glucuronoxylans during the kraft pulping process was investigated. Woods and pulps xylans were isolated and characterized by size exclusion chromatography, methylation (linkage) analysis and ¹H NMR. Eucalyptus globulus and Eucalyptus urograndis showed xylan retention significantly higher than that of Betula pendula. The higher retention of Eucalyptus xylans was assigned to (i) their higher average molecular weight (31 against 24 KDa in B. pendula) and to (ii) the presence of O-2 substituted 4-O-methyl--d-glucuronic acid groups ([→2)-GlcpA-(1→]) with galactopyranosyl/glucopyranosyl residues belonging to fragments of galactan/glucan chains that were absent in B. pendula xylans. A significant part of uronic acids, particularly [→2)-GlcpA-(1→] units, remain in fibres until the end of pulping. The acetylation degree and distribution of acetyl groups between Xylp units, in general terms, was similar in the three types of xylans. Unexpectedly, about 20% of the acetyl groups persisted in pulps xylans till the end of pulping.     

Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent K _m value for birchwood xylan was much lower than the K _m value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain. Received: 6 May 1998 / Accepted: 29 July 1998     

Degradation of lignocellulose by extracellular enzymes produced by Thermomonospora fusca BD25

Degradation products from the addition of extracellular enzymes from Thermomonospora fusca BD25 to ball-milled wheat straw, oat spelt xylan and solubilised kraft pulps were characterised by HPLC and TLC. Overall, a high percentage hydrolysis of oat spelt xylan (28.9%) occurred after 26 h incubation. However, the rates of hydrolysis of ball-milled wheat straw and kraft pulp were approximately 4-6-fold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate was similar (22.2% and 25.9% respectively). Incubation of kraft pulp and ball-milled wheat straw by crude extracellular enzymes of T. fusca BD25 resulted in the detection of aromatic compounds at concentrations of 0.6 microg ml(-1) and 8.7 microg ml(-1), respectively. Hydrolysis of oat spelt xylan by T. fusca BD25 extracellular enzymes yielded a mixture of xylose, xylotriose and putative substituted-xylotriose, while the products of ball-milled wheat straw hydrolysis were xylose, glucose and a small oligomer present in the digest. The results highlight the ability of culture supernatant from T. fusca to release both simple sugars and aromatic compounds from lignocellulosic substrates and suggest a role for this organism in the biobleaching of pulp.     

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     

Substrate hydrolysis by combinations of Trichoderma xylanases

The hydrolysis of five xylan substrates was examined using combinations of two pairs of xylanases from two species of Trichoderma. Antisynergy was observed in acetylated xylan isolated from aspen when the maximum hydrolysis achieved by certain xylanase combinations was significantly lower than that achieved by the most effective enzyme in the combination. Cooperative interactions among xylanases were observed in pine holocellulose where xylanase combinations were more effective than single xylanases.     

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities.

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.     

Evaluation of hydrolysis products of xylans by xylan-degrading enzymes from Humicola grisea var. thermoidea and Aspergillus fumigatus Fresenius

Xylan-degrading enzyme activities were isolated from crude extracts of solid-state cultures of Aspergillus fumigatus Fresenius (Xyl I, Xyl III, Xyl IV and Xyl V) and Humicola grisea var. thermoidea (Xyl II) by chromatographic procedures. The pattern of hydrolysis of different xylans and pulps varied from traces of xylose to xylooligomers. The products formed suggest an endo-type enzyme mode of action. Some enzymes showed debranching and transglycosidase activities.     

Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).     

Relationships between activities of xylanases and xylan structures

Structures of five water-soluble xylans have been determined. Four purified xylanase enzymes have been studied for the hydrolysis of the xylans. Different xylanases have different activities against various xylan structures. The key factors that influence the rate of xylan hydrolysis are chain length and degree of substitution. Two family 11 xylanases, Orpinomyces pc2 xylanase and Trichoderma longibrachiatum xylanase, can rapidly hydrolyze xylans that have a chain length greater than 8 xylose residues, and their hydrolytic rates are not sensitive to substituents on the xylan backbone. A family 11 xylanase from Aureobasidium pullulans is most effective on xylans that have a long chain (greater than 19 xylose residues), and also is effective against substituent groups. Although Thermatoga maritima xylanase is also more active on a long xylan chain (greater than 19 xylose residues), its hydrolytic rate is greatly reduced by substituents on xylan backbones.     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

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.     

Effect of Wood Modification on Lignin Consumption and Synthesis of Lignolytic Enzymes by the Fungus Panus (Lentinus) tigrinus

Lignin consumption and synthesis of lignolytic enzymes by the fungus Panus (Lentinus) tigrinuscultivated on solid phase (modified and unmodified birch and pine sawdusts) were studied. The fungus grew better on and consumed more readily the birch lignin than the pine wood. Peroxidase activity was higher in the case of pine sawdust; laccase and lignolytic activities, in the case of birth sawdust. Treatment with ammonia or sulfuric acid decreased lignin consumption by this fungus cultivated on either medium. Modification of sawdust by ultrasound increased lignin consumption and may be recommended for accelerating biodegradation of lignocellulose substrates.     

Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi

A beta-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35 degrees C, with a secondary structure under those conditions that consists of approximately 10 to 15% alpha-helices. The enzyme was still active at temperatures higher than 40 degrees C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45 degrees C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-D-glucuronic acid and appears to require two 4-O-methyl-D-glucuronic acid residues for substrate recognition and/or cleavage of a beta-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process.