The non-cellulolytic Thermomyces lanuginosus is a widespread and frequently isolated thermophilic fungus. Several strains of this fungus have been reported to produce high levels of cellulase-free β-xylanase both in shake-flask and bioreactor cultivations but intraspecies variability in terms of β-xylanase production is apparent. Furthermore all strains produce low extracellular levels of other hemicellulases involved in hemicellulose hydrolysis. Crude and purified hemicellulases from this fungus are stable at high temperatures in the range of 50–80°C and over a broad pH range (3–12). Various strains are reported to produce a single xylanase with molecular masses varying between 23 and 29 kDa and pI values between 3.7 and 4.1. The gene encoding the T. lanuginosus xylanase has been cloned and sequenced and is shown to be a member of family 11 glycosyl hydrolases. The crystal structure of the xylanase indicates that the enzyme consists of two β-sheets and one α-helix and forms a rigid complex with the three central sugars of xyloheptaose whereas the peripheral sugars might assume different configurations thereby allowing branched xylan chains to be accepted. The presence of an extra disulfide bridge between the β-strand and the α-helix, as well as to an increase in the density of charged residues throughout the xylanase might contribute to the thermostability. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase has made the fungus an attractive source of thermostable xylanase with potential as a bleach-boosting agent in the pulp and paper industry and as an additive in the baking industry.
Thermomyces lanuginosus (formerly known as Humicola lanuginosa) is a widely distributed thermophilic fungus commonly isolated from self-heating masses of organic debris . It was first isolated in 1899 by Tsiklinskaya from a potato, which had been inoculated with garden soil and grown on white bread kept at 52–53°C . By definition, a thermophilic fungus is one that thrives at temperatures up to 60°C and fails to grow below 20°C . A further characteristic feature of thermophiles is that their enzymes are more heat stable than those of mesophiles when extracted and tested in cell-free systems .
Several strains of T. lanuginosus have been found to be hyperproducers of extracellular xylanase but other hemicellulases are also produced. These enzymes are responsible for hydrolysis of hemicellulosic materials, which are heteropolysaccharides found in association with cellulose constituting about 20–30% of the wood dry weight . Microbial xylanases (1,4-β-D-xylan xylanohydrolase, EC 18.104.22.168) have been the most widely studied of all hemicellulases and are the preferred catalysts for hydrolysis of xylan, the most common hemicellulosic polysaccharide, due to their high specificity, mild reaction conditions, negligible substrate loss and minimal side product formation .
Crude and purified xylanases and several other hemicellulases produced by most strains of T. lanuginosus are stable at very high temperatures and over a broad pH range [7–14]. The use of hemicellulolytic enzymes, xylanases in particular, has attracted considerable interest by the pulp and paper industry to eliminate the use of chlorine chemicals in pulp bleaching [14–17]. Although T. lanuginosus produces high levels of xylanases, several studies have shown that different strains of this fungus vary in their expression of xylanases, mannanases and other glycosyl hydrolases. Apart from xylanase, most of the glycosyl hydrolases are expressed at very low levels [8,11–13,18–24]. Growth on a variety of carbon and nitrogen sources has also been shown to affect enzyme production by different strains of T. lanuginosus[8,11–13,18–24]. The ability of T. lanuginosus to produce high levels of cellulase-free thermostable xylanase is attractive to those considering biotechnological application of the enzyme [18–24].
T. lanuginosus also produces a range of other secreted degradative enzymes such as α-amylase , glucoamylase , pectinase , phytase , protease  and lipase . These enzymes have been studied to a limited extent and are reported to be thermostable catalysts. The reader is referred to a recent review where details of these enzymes are presented .
This article presents an overview of current research on the properties of the fungus T. lanuginosus and production, characteristics and application of its hemicellulases.
2Occurrence, morphology, properties and taxonomic relationships
T. lanuginosus occurs worldwide and has been isolated in many countries from a variety of decaying plant material [8,11,12,14,18,22,23,30]. It is a thermophilic fungus, as strains generally grow between a maximum temperature of 60°C and a minimum of 20°C with an optimum growth temperature of 50°C. By comparison, thermotolerant fungi generally have a growth temperature maximum of about 50°C and a temperature minimum well below 20°C whereas the growth temperature maximum of mesophilic fungi is generally below 37°C (Fig. 1) . The optimum growth pH of most strains investigated is 6.5. T. lanuginosus strains have been reported to occur in dry and waterlogged grassland, loamy garden soil and aquatic sediments but the fungus is more specifically associated with organic substrates such as the culms, roots and leaves of grasses, composts of various plant materials and the dung of various birds and mammals [1,2,8,11,12,14,22,23,30]. It has even been isolated from air in Indonesia and the British Isles . The reader is referred to the websites of the American Type Culture Collection (http://www.atcc.org) and the Centraalbureau voor Schimmelculturen (http://www.cbs.knaw.nl) for more details on the geographic origins of the strains.
Since T. lanuginosus is non-cellulolytic, it probably grows commensally in composts with cellulolytic fungi by utilising some of the sugars generated by the enzymes of these fungi and possibly by also using their mycelial breakdown products . T. lanuginosus may also rely upon cellulolytic fungi as a source of nitrogen as they release a number of amino acids into their environment. In the absence of cellulolytic fungi, T. lanuginosus is commonly associated with Mucor pusillus, as both fungi utilise similar simple carbon sources .
T. lanuginosus is classified as a Deuteromycete (imperfect fungus), that is unicellular or septate and reproduces asexually by forming aleurioconidia . On various media, colonies of T. lanuginosus grow rapidly, reaching 2.5 to over 5 cm in diameter at 45–50°C within 2 days. Initially the colonies appear white and felt-like and are less than 1 mm high, but soon turn grey or greenish-grey, commencing from the centre of the colony. Subsequently the colony turns purplish brown and the agar substratum stains a deep pink or wine colour, due to diffusible substances secreted by the colony. Mature colonies appear dull dark brown to black (Fig. 2) .
Immature conidia with a diameter of 5.5–12 μm are colourless and smooth walled. They turn dark brown and globose as they mature and form a thick outer conidial wall that is characteristically wrinkled (Fig. 2) . Aleuriospores are generally unbranched but occasionally they branch once or twice near the base and appear to cluster. Septations commonly occur in the aleuriospores but they are difficult to observe. The mycelium is partly found on the surface, and partly immersed and there are no stroma or hyphopodia. The aleuriospores are straight or curved, colourless or brown and smooth .
Hemicelluloses are heterogeneous polysaccharides, which are located between the lignin and cellulose fibres and, depending on wood species, constitute about 20–30% of the naturally occurring lignocellulosic plant biomass . Hemicelluloses are composed of both linear and branched heteropolymers of D-xylose, L-arabinose, D-mannose, D-glucose, D-galactose and D-glucuronic acid, which may be acetylated or methylated. Most hemicelluloses contain two to six of these sugars . They are usually classified according to the main sugar residues in the backbone . The two main hemicelluloses in wood are xylans and glucomannans, both of which are present in softwood whereas in hardwood, xylan is the main hemicellulose component .
Xylan is the most abundant hemicellulosic polysaccharide, comprising up to 30% of the cell wall material of annual plants, 15–30% of hardwoods and 7–10% of softwoods . Xylan occurs as a heteropolysaccharide with a homopolymeric backbone chain of 1,4-linked β-D-xylopyranose units, which consists of O-acetyl, α-L-arabinofuranosyl, α-1,2-linked glucuronic or 4-O-methylglucuronic acid substituents. In hardwoods, xylan exists as O-acetyl-4-O-methylglucuronoxylan with a higher degree of polymerisation (150–200) than softwoods (70–130), which occurs as arabino-4-O-methylglucuronoxylan . Approximately one in 10 of the β-D-xylopyranose backbone units of hardwood xylan are substituted at C-2 position with 1,2-linked 4-O-methyl-α-D-glucuronic acid residue, whereas 70% are acetylated at C-2 or C-3 positions or both . Softwood xylans are not acetylated but the 4-O-methylglucuronic acid and the L-arabinofuranose residues are attached to the C-2 and C-3 positions, respectively of the relevant xylopyranose backbone units .
The xylan of grasses occurs as arabino-4-O-methylglucuronoxylan with a degree of polymerisation of 70 and contains L-arabinofuranosyl side chains linked to C-2 or C-3 positions, or both, of the β-D-xylopyranose main chain residues . The 1,2-linked 4-O-methyl-α-D-glucuronic acid is found less frequently in grasses than in hardwood xylan. Hardwood xylan contains 2–5% by weight of O-acetyl groups linked to C-2 or C-3 positions of the xylopyranose units, and 6% of the arabinosyl side chains are themselves substituted at position 5 with feruloyl groups, whereas 3% are substituted with p-coumaroyl residues. The relative proportions of the various components of grass arabinoxylan vary from species to species and from tissue to tissue within a single species .
The main mannan group in the cell walls of higher plants is galactoglucomannan with the backbone composed of 1,4-β-linked D-glucose and D-mannose units that are distributed randomly within the molecule. Softwood glucomannan has a backbone composed of β-1,4-linked D-glucopyranose and D-mannopyranose units, which are partially substituted by α-galactose units and acetyl groups . Hardwoods contain a small amount of glucomannan (2–5%) that has no galactose or acetic acid side groups present .
Due to the complex structure of hemicelluloses, several different enzymes are needed for their enzymatic degradation or modification. The two main glycosyl hydrolases depolymerising the hemicellulose backbone are endo-1,4-β-D-xylanase and endo-1,4-β-D-mannanase . Since xylan is a complex component of the hemicelluloses in wood, its complete hydrolysis requires the action of a complete enzyme system (Fig. 3), which is usually composed of β-xylanase, β-xylosidase, and enzymes such as α-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase, and hydroxycinnamic acid esterases that cleave side chain residues from the xylan backbone. All these enzymes act cooperatively to convert xylan to its constituents .
Xylanases attack randomly the backbone of xylan to produce both substituted and non-substituted shorter chain oligomers, xylobiose and xylose . Xylosidases are essential for the complete breakdown of xylan as they hydrolyse xylooligosaccharides to xylose . The enzymes arabinosidase, α-glucuronidase and acetylxylan esterase act in synergy with the xylanases and xylosidases by releasing the substituents on the xylan backbone to achieve a total hydrolysis of xylan to monosaccharides .
β-Mannanases catalyse the random hydrolysis of β-D-1,4-mannopyranosyl linkages within the main chain of mannan and various polysaccharides consisting mainly of glucomannan, galactomannan and galactoglucomannan . Other glycosyl hydrolases that are important for the degradation of mannan include mannosidases and galactosidases. β-Mannosidase catalyses the hydrolysis of terminal, non-reducing β-D-mannose residues in mannan [5,34]. α-Galactosidases hydrolyse terminal, non-reducing α-D-galactosides from galactose oligosaccharides, galactomannan and galactoglucomannan and are also capable of removing α-1,6-bound galactosyl units from polymeric galactomannan .
5Production of hemicellulases
The majority of microorganisms growing on plant residues in nature usually produce both cellulolytic and xylanolytic enzymes due to the close association of cellulose and xylan in plant cell walls. However, a number of microorganisms are only able to degrade xylan . T. lanuginosus has been shown in numerous studies to produce extremely high levels of xylanase but not to produce cellulose-degrading enzymes [8,12,20].
Efficient production of xylanolytic enzymes is dependent upon the choice of an appropriate inducing substrate and the medium composition . Generally, xylanases are induced in most microorganisms during growth on substrates containing xylan . Xylan, being a high molecular mass polymer, cannot penetrate the cell wall and apparently low molecular mass fragments of xylan play a key role in the regulation of xylanase biosynthesis. These fragments include xylose, xylobiose, xylooligosaccharides and heterodisaccharides containing xylose that are liberated from xylan by the action of low levels of constitutively produced enzymes . Xylanase production by T. lanuginosus was shown to correspond to an induction repression mechanism. A low basal level of xylanase is constitutively formed without the presence of an inducing substance. In the presence of D-pentoses xylanase production is induced in T. lanuginosus DSM 5826 with D-xylose having the strongest effect indicating that D-xylose was the natural inducer . In the presence of easily metabolisable substances such as glucose, fructose or lactose, xylanase is formed, although the activity in the presence of these repressors is similar to basal levels . Xylan had the most pronounced effect on xylanase production by this fungus as the level of induction by D-xylose, D-arabinose, D-ribose, and L-arabinose does not occur to the same degree as when xylan is used as substrate. During the initial induction period, T. lanuginosus DSM 5826 only formed constitutive levels of xylanase activity, which led to slow liberation of xylooligosaccharides from xylan. These fragments induce xylanase production, thus leading to a higher final level of enzyme activity. The reason for this effect is believed to be the longer availability of inducing molecules that are slowly liberated, thus resulting in a delayed exhaustion of an inducer. A similar mechanism was also suggested by Biely  to explain the induction of xylanase in the yeast Cryptococcus albidus by xylobiose. With the sequential addition of xylose, xylanase formation was delayed but lasted longer. The kinetics of xylanase secretion shows a dependence on the concentrations of the inducer. Therefore the availability of an inducer at low levels over an extended period was thought to lead to hyperproduction of enzyme in T. lanuginosus DSM 5826 .
5.1Hemicellulase production in shake-flask cultivations
T. lanuginosus strain SSBP has thus far been reported to be the best producer of cellulase-free crude xylanase when grown on coarse corncobs with an activity of 3575 U ml−1 and a specific activity of 3005 U mg−1 (Table 1). These levels were much higher than xylanase activity levels of 2172 and 2726 U ml−1 produced by T. lanuginosus strains DSM 5826 and ATCC 46882, respectively, grown on the same substrate in shake-flask cultures [8,18,20,21]. When T. lanuginosus was cultivated on various carbon sources (31.2 g l−1), significant differences in xylanase production occurred and with the exception of xylose, other easily metabolisable hexoses and pentoses failed to induce high levels of xylanase [8,19,20]. Corncobs were found to be the most effective substrate for xylanase production among various lignocellulosic substrates evaluated, such as corn leaf, wheat bran, wheat straw, barley husk and birchwood xylan [8,10,11,19,21]. The high xylanase production on coarse corncobs may be due to their greater particle size (coarse corncobs, 2–7 mm; fine corncobs <2 mm), which leads to slower solubilisation of reducing sugars and creates a support system for fungal growth and enzyme release . The effect of various organic nitrogen compounds on the production of xylanase by T. lanuginosus strains showed that all sources promoted growth of the fungus, but yeast extract had the most pronounced effect (Table 2). In an attempt to distinguish between high and low xylanase producing strains, Singh et al.  examined the phylogenetic properties of eight strains. While no differences were found in the sequence of the internal transcribed region of the 5.8S rDNA, random amplified polymorphic DNA (RAPD) analyses showed that a relationship between the RAPD pattern and levels of xylanase produced could be established using certain primers. This observation would assist in attempts to find other high xylanase producing strains.
Table 1. Effect of carbon sources on xylanase production by some strains of T. lanuginosus
Evaluation of the production of other hemicellulases such as xylosidase, arabinosidase, acetyl esterase, acetyl xylan esterase, feruloyl esterase, and ρ-coumaroyl esterase by T. lanuginosus revealed that complex growth substrates such as xylan, corncobs, wheat bran and locust bean gum induced only low enzyme levels (<1 U ml−1) [8,20]. Mannanase and mannosidase were not detected in T. lanuginosus strain DSM 5826 after growth on corncobs whereas both enzymes were produced at low levels in T. lanuginosus SSBP (<2 U ml−1). However, growing the fungus on galactomannan (locust bean gum) did not result in the induction of higher levels of mannan-degrading enzymes than other xylan-containing substrates . Furthermore, Puchart et al.  reported that only three of the 17 T. lanuginosus strains (IMI 158749, CBS 218.34 and IMI 131010) tested were able to produce mannanase on locust bean gum; hence mannanase production is apparently strain dependent . However, there appears to be some doubt concerning the identity of the best mannanase producing strain (IMI 158749) and this strain might be reclassified on the basis of rDNA analysis (P. Biely, B.A. Prior and Z. Ögel, unpublished data).
The influence of agitation on xylanase production of T. lanuginosus DSM 5826 was investigated . A shaking speed of 120 rpm provided the optimal conditions for enzyme formation. At a decreased shaking speed of 100 rpm, the fungus showed poor growth and enzyme production was reduced dramatically whereas at higher shaking speeds of 150–250 rpm enzyme production was adversely affected. The lower xylanase activity produced at the slower shaking speed was ascribed to poor oxygen transfer within the medium, whereas the lower xylanase production at higher shaking speeds was thought to be due to greater hyphal branching, mycelial fragmentation and early sporulation .
5.2Hemicellulase production in bioreactor cultivations
The agitation rate, dissolved oxygen tension (DOT) and aeration rates have been shown to influence enzyme productivity in a bioreactor. However optimal conditions are apparently unique for each microbial strain and process and it is essential to evaluate the effects of aeration rates on enzyme production by microorganisms, especially filamentous fungi that are sensitive to shear . Shear stress within the medium, which is directly related to the stirrer speed, has a marked influence on xylanase production by T. lanuginosus SSBP [8,24]. The sensitivity to shear stress has also been reported for other filamentous fungi. The production of cellulase and xylanase by Aspergillus fumigatus and Aspergillus awamori is negatively influenced by the rate of stirring and the lower production at high shear rates could be caused by hyphae disruption .
Studies on xylanase production in a bioreactor have revealed a significant increase in xylanase levels of T. lanuginosus strain SSBP grown on xylose compared to shake-flasks (Table 3) [24,41]. Rheological problems were encountered when coarse corncobs were used as the carbon source for xylanase production in a 30-l bioreactor . In other studies it was reported that xylanase levels were lower in bioreactors than in shake-flasks when T. lanuginosus strains DSM 5826 and RT 9 were cultivated on xylan substrates [11,12,23,39].
Table 3. Production of β-xylanase in bioreactors by T. lanuginosus strains
Purkarthofer et al.  reported that both the culture pH value and stirrer speed strongly affect xylanase formation by T. lanuginosus strain DSM 5826. At a stirrer speed of 50 rpm and a controlled pH value of 7.5, T. lanuginosus produced the highest xylanase activity of 1950 U ml−1. However, in another study with the same fungus, low xylanase activity (427 U ml−1) produced was ascribed to pH control over the duration of the cultivation and the requirement for high stirrer speed, necessary to maintain dissolved oxygen concentrations above 5%. In a further bioreactor study it was revealed that T. lanuginosus strain RT 9 had an optimum stirring speed of 200 rpm and an increase in the oxygen transfer rate appeared to increase xylanase production . Xylanase production by T. lanuginosus strain SSBP grown on xylose was also adversely affected by agitation . A maximum xylanase activity of 260 U ml−1 was reached after 18 h at 1000 rpm and an increase in stirrer speed to 1300 rpm did not enhance any further xylanase production. Maximum xylanase production on xylose occurred at a lower stirrer speed of 400 rpm, even though the DOT decreased during active growth, reaching less than 1% after 20 h. This clearly demonstrated the interrelationship between agitation rate and DOT and their influence on xylanase production .
The levels of other hemicellulose-degrading enzymes produced in bioreactors by several T. lanuginosus strains were low compared to xylanase production [11,12,23,24,39,41]. Growth conditions, however, also affect the levels produced. For example, when T. lanuginosus strain SSBP was grown on xylose, xylosidase levels were higher in the bioreactor cultures than in shake-flask cultures . Furthermore, differences in levels of other hemicellulases produced were related to the T. lanuginosus strain and the aeration and agitation rates [23,24,41].
6Thermal stability of hemicellulases
The half-life (T1/2) of enzyme activity is generally regarded as the most accurate and reliable way to evaluate the thermostability of an enzyme. Enzyme thermostability is also influenced by protein concentrations in the culture supernatant. This factor as well as different experimental approaches may contribute to variations in results reported by several investigators on the same strains [7,11,12]. Xylanases produced by thermophilic fungi are usually more thermostable than those of mesophilic fungi [42,43]. Furthermore, xylanases produced by thermophilic eubacteria and archaea have considerably longer T1/2 at 80°C or higher temperatures than those from thermophilic fungi . However, the levels of xylanase produced by these bacteria are considerably lower than those of fungi.
T. lanuginosus has been reported by a number of laboratories to produce a highly thermostable xylanase based on the T1/2 of the enzyme [7,9,11,12,41]. The xylanase of T. lanuginosus strain SSBP was reported to be the most stable (T1/2= 337 min at 70°C), whereas that of the DSM 5826 strain (T1/2= 201 min at 70°C), and other strains showed lesser degrees of stability (T1/2= 126 min or less at 70°C). The xylanase of T. lanuginosus strain SSBP retained its full activity at temperatures up to 65°C and 45% of its activity after 30 min at 100°C and also retained its total activity after 14 days at 60°C . The xylanases from other strains demonstrated a lower degree of enzyme activity and the xylanase produced by the strain ATCC 16455 was the least stable with only 10% activity remaining after 30 min at 100°C. In contrast, the xylanase of T. lanuginosus strain RT 9 had previously been reported to retain its full activity at temperatures of up to 80°C after 30 min and 68% of its activity after 30 min at 100°C . However, a 48% loss was observed for the storage stability tests at 55°C after 21 days. The other hemicellulases produced by T. lanuginosus strain SSBP were much less thermostable than the xylanase in the temperature range of 40–60°C .
6.2Influence of various factors on thermostability
Xylanases from various strains were stable (maintain 90% of activity over 30 min) over a wide range of pH values (3–11 at 20°C and less) but the pH stability apparently narrowed (5.5–9.5) with increase in temperature (70°C) [7,8,11–13]. Furthermore, at 70°C the xylanase from strain SSBP had a broader range of pH stability than that of strain DSM 5826. The pH range of the other hemicellulases from strain SSBP at which they were thermostable was narrower in most instances  than that reported for the xylanase [7,8,11–13].
In the presence of sorbitol (50% w/v), and glycerol (50% v/v), the xylanase from T. lanuginosus strain SSBP retained 100 and 90% activity after 6 h incubation at 70°C, suggesting that these polyhydric alcohols could be useful in situations were greater enzyme thermostability is required .
The type of carbon source used for the growth of T. lanuginosus has also been reported to affect the thermostability of its xylanases . The xylanase produced by T. lanuginosus strain SSBP on coarse corncobs was the most thermostable. Surprisingly, the activity of the enzyme increased by 6% when the temperature was raised from 70 to 80°C whereas the thermostability of the xylanase produced on other substrates decreased as the temperature was increased . When the culture supernatant from corncob-grown cells was dialysed, the thermostability increase disappeared and the xylanase was much less thermostable suggesting the presence of an unknown compound . Gomes et al.  also reported differences in thermostability of T. lanuginosus xylanases produced on various lignocellulosic substrates with the xylanases produced on corncobs and corn leaf as the most thermostable.
7Biochemical properties of purified hemicellulases
Xylanase of T. lanuginosus has been purified from a number of strains (Table 4). The molecular mass of the enzyme was found to be in the range of 22.5–29.0 kDa depending on the method of determination and apparently is not glycosylated. The pI value of the xylanase from various strains was reported to be between 3.8 and 4.1 [21,30,45–47]. Furthermore, in a carefully controlled study on eight strains grown on corncobs, the analysis of the xylanase in the culture supernatant by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and isoelectric focusing revealed the presence of a single enzyme of 24.7 kDa and pI 3.9 . Xylanases can be differentiated into two families (10, formerly family F; and 11, formerly family G) on the basis of their molecular mass, pI values, hydrophobic cluster analysis and amino acid sequence homologies . Family 10 xylanases have a high molecular mass/low pI value whereas family 11 xylanases have low molecular mass/high pI value. Greater catalytic versatility is found in family 10 than in family 11 xylanases whereas family 10 xylanases hydrolyse naturally occurring polysaccharides to a greater extent . The xylanase of T. lanuginosus is apparently uncharacteristic as it is a low molecular mass/low pI value enzyme. Xylanase from strains ATCC 46882 and SSBP liberated mainly xylose and xylobiose from beechwood O-acetyl-4-O-methyl-D-glucuronoxylan [21,46]. Similarly xylanase from strain ATCC 46882 released xylose and xylobiose from beechwood 4-O-methyl-D-glucuronoxylan and rhodymenan (β-(1→3)-β-(1→4)-xylan) and in addition also released an acidic xylooligosaccharide from 4-O-methyl-D-glucuronoxylan and isomeric xylotetraose and xylopentaose from rhodymenan. The molecular mass of the xylanase and the nature of the fragments released by the xylanase on various substrates point to the enzyme belonging to family 11. Furthermore Schlacher et al.  found that the amino acid sequence of the xylanase from strain DSM 5826 was highly homologous to other family 11 xylanases. Based on the evidence above it is apparent that the xylanase of T. lanuginosus strains belongs to family 11.
Table 4. Biochemical properties of purified xylanases produced by T. lanuginosus strains
The optimum temperature and pH of purified xylanase from various strains have been reported to be in the range of 60–75°C and 6.0–7.0 respectively (Table 4). These values are similar to those observed in crude extracts of xylanase (7–9, 11–13). The temperature stability of the purified xylanase from various strains differed somewhat depending on the experimental conditions (Table 4). Overall the crude enzyme (T1/2= 337 min at 70°C) is apparently more thermostable than the purified xylanase of T. lanuginosus strain SSBP (retained only 20% activity at 75°C after 30 min)  suggesting that some unknown factors might be present in the extract that stabilise the protein. The kinetic properties of only two purified xylanases from T. lanuginosus have been investigated (Table 4).
Only one other hemicellulose-degrading enzyme from T. lanuginosus has been purified and characterised. A purified extracellular α-galactosidase from T. lanuginosus strain IMI 158749 was found to have a molecular mass of 57 kDa and an isoelectric point of 5.2 . The enzyme had optimum α-galactosidase activity at 65°C and pH 4.5–5.0 and was stable for 6 h at 60°C and at pH 3.0–7.5, with a half-life of 3 h at 65°C. This enzyme was most active against aryl α-D-galactosides but also could efficiently hydrolyse α-glycosidically linked non-reducing terminal galactopyranosyl residues such as melibiose, raffinose, stachyose, and fragments of galactomannan occurring in natural substrates.
8Molecular and structural biology of xylanase
A detailed structural and sequence investigation of enzymes from thermophilic fungi such as T. lanuginosus has allowed the essential properties of these enzymes to be identified . The cDNA and genomic DNA fragments encoding the thermostable xylanase from strain DSM 5826 have been cloned and sequenced . The cDNA sequence revealed an open reading frame encoding a polypeptide of 225 amino acids that corresponds to a calculated molecular mass of 21.314 kDa. A typical putative signal sequence is represented by the N-terminal 19 amino acids. A putative KEX-like protease cleavage site is apparent in the region around the amino acid residue at position 41 (Fig. 4) and results in a processed polypeptide starting at the amino acid glutamine. Comparison of the cDNA with the genomic DNA sequence showed that the xylanase was encoded by two exons interrupted by a 106 bp long intron, showing perfect 5′ (AGBGTANGT) and 3′ (ACAGB) intron splice sites with the internal consensus sequence ‘CAGCTAAC’. The conserved sequence TATAAA and CCAAT motif, which are commonly found in eukaryotic promoters were found upstream of the putative translation initiation ATG codon (89 and 202 bp respectively). Comparison of T. lanuginosus xylanase to other low molecular mass fungal xylanases showed homology between 52 and 67% sequence identity with 44 amino acid residues strictly conserved (Fig. 4). A comparison of fungal and bacterial xylanases shows a much lower degree of conservation of amino acid residues . The catalytically active site of the T. lanuginosus xylanase possesses two strictly conserved glutamate residues (positions 129 and 226, Fig. 4) . The region surrounding these residues is highly conserved in the fungi. Among all family 11 xylanases the first glutamate residue is highly conserved, whereas the other shows variability, which may account for the different pH stability values found with these enzymes . There are also two non-conserved cysteine residues in positions 153 and 202 (Fig. 4) which form a disulfide bridge, not found in the majority of other family 11 xylanases and have been suggested by Schlacher et al.  to be responsible for the high thermostability of the T. lanuginosus xylanase. Surprisingly these two cysteine residues are also present in the xylanase of the mesophilic fungus Schizophyllum commune (Fig. 4), implying that other factors in addition to the cysteine residues confer thermostable properties .
The xylanase from strain DSM 5826 has been crystallised and found to be a compact, globular protein with a long cleft spanning the whole molecule, containing the active site and dominated by two twisted β-sheets . The two β-sheets and the α-helix form the ‘fingers’ and the ‘palm’ of a right hand whereas the two loop regions form the ‘thumb’ and a ‘cord’. In these loop regions, most of the deletions and insertions are found. The disulfide bridge connects the C-terminus of a β-sheet with the N-terminus of the α-helix. An extension of an existing network of charged residues of the enzyme stabilises the most sensitive region of the molecule .
To our knowledge, no other hemicellulases have been cloned from T. lanuginosus although genes encoding other proteins such as kinesins, actin and phytase have been cloned and characterised [26,53–55].
Xylanase genes from various microbial genera have been heterologously expressed in Escherichia coli but invariably the expression level has been lower than that found in the parent organism as the enzyme is confined to either the cytoplasmic or the periplasmic fraction . The absence of post-translational modifications such as glycosylation in E. coli and the intracellular accumulation of the recombinant xylanases have been suggested to be the key reasons for low extracellular enzyme activities. The xylanase gene incorporating the secretion signal sequence of T. lanuginosus strain DSM 5826 was functionally expressed in E. coli as a Lac Z-fusion protein but extracellular enzyme activity was not reported . Subsequently the recombinant E. coli strain was found to produce up to 240 U ml−1 of intracellular xylanase when induced with 0.1 mM isopropyl thiogalactoside but with very low extracellular levels (D. Stephens, K. Rumbold, B.A. Prior and S. Singh, unpublished results). However, the phytase gene from T. lanuginosus has been expressed under transcriptional control of the Fusarium oxysporum trypsin gene promoter in Fusarium venenatum. In this heterologous host, the recombinant enzyme was secreted without apparent intracellular accumulation. The phytase was enzymatically active between pH 3 and 7.5, retained activity at assay temperatures up 75°C and demonstrated superior catalytic activity to any known fungal phytase at 65°C. Comparison of this Thermomyces catalyst with the well-known Aspergillus niger phytase reveals other favourable properties including catalytic activity over an extended pH range .
9Biotechnological applications of hemicellulases
Hemicellulases, especially xylanases, have been applied in processes such as pulp bleaching, baking, clarification of juices, extraction of coffee, plant oils and starch and as a feed supplement to improve digestion in animals. Other potential applications include the conversion of xylan in wastes from agricultural and food industry into xylose, and the production of fuel and chemical feedstocks .
In pulp bleaching, xylanases selectively degrade the accessible hemicellulose fraction of woods and have been found to enhance the extractability of lignin . Xylanases from several T. lanuginosus strains have exhibited promising results when applied as a bleaching agent to kraft and sulfite pulp produced from sugar cane bagasse, Eucalyptus and beech. Significant reduction of the use of bleaching chemicals required to attain the desired kappa number was found while increased brightness and viscosity was achieved [14–17,57]. Commercial xylanases are typically produced by mesophilic filamentous fungi such as Trichoderma reesei and A. niger, which are excellent protein secretors. However, these xylanases may not be sufficiently thermostable for processes where enzymes active at higher temperatures have a competitive advantage. Therefore the thermostable xylanase of T. lanuginosus may be suitable for such high temperature processes. Deinking of laser-printed paper was achieved when treated individually, and with combinations of a xylanase from strain DSM 5826, a mannanase from S. rolfsii and purified endoglucanases from Gloeophyllum sepiarium and Gloeophyllum trabeum. The enzyme treatment in these studies was conducted at a temperature more appropriate for the optimum temperature of the enzymes from the mesophilic fungi than the xylanase from the thermophilic fungus T. lanuginosus. However, the combination of enzymes resulted in 50% more mannan and 11% more xylan being solubilised than did the individual enzymes and illustrated the synergistic effects of using enzymes together for treatment of pulp.
Addition of xylanase at the correct dosage to cereal-based foods such as bread, pasta and noodles yields more flexible, easy-to-handle dough thereby improving the final baked product. A baking process based on the use of xylanolytic enzymes from T. lanuginosus strains has been patented and was reported to improve the baking properties of dough . Hemicellulases, in addition to cellulases, also play an important role in the digestion of grass and hay by ruminants and a xylanase from T. lanuginosus was shown to improve in vitro rumen degradation of wheat straw when used as a supplement (F.M. Lakay, W.H. van Zyl and B.A. Prior, 2000, unpublished results). Xylanases are also used in poultry nutrition in order to minimise problems with droppings  but no reports of the use of T. lanuginosus xylanases for this purpose are documented.
10Concluding remarks and perspectives
Extensive investigations of T. lanuginosus have shown that this thermophilic fungus possesses a number of exceptional properties. The ability of a number of strains to produce high levels of thermostable xylanase is notable and is comparable to the best xylanase producing organisms yet reported. However, strains show significant variability in their ability to produce xylanase and this points to considerable genetic diversity in this species. This apparent diversity was found in RAPD analysis but not the sequence of the internal transcribed region of the 5.8S rDNA. More extensive studies on the molecular, physiological and ecological properties of T. lanuginosus strains are apparently justified. Furthermore, the strains are also influenced significantly by the nutrient and growth conditions used in their cultivation and this implies that in order to achieve maximal xylanase production, the cultivation conditions of each strain will require optimisation.
In light of the exceptionally high levels of extracellular xylanase produced by T. lanuginosus, it is surprising that such low levels of other hemicellulases are found in the culture fluids of this fungus and that cellulose is not attacked. This indicates that the organism might require the synergistic activities of other organisms in order to degrade lignocellulose in the environments where it is commonly found. It is also not clear how the organism completes hemicellulose degradation. Whether the oligosaccharide breakdown products are degraded further on the surface of the fungus or intracellularly has apparently not been investigated.
There is a lack of clarity with regard to the basis of the thermostability of the xylanase. In some reports, unknown extracellular components apparently contributed to the thermostability but the nature of these factors is unknown. Whether there are subtle differences in the amino acid sequences that could influence the thermostability of the xylanase has also not been reported as the xylanase gene from only one strain has been sequenced to date.
The xylanase of T. lanuginosus has considerable biotechnological potential. With the recent proclamation of stringent environmental regulations in many countries, it has become important to develop processes that are environmentally safe and at the same time cost effective. The use of xylanase in the bleaching of wood pulp is an attractive alternative to the chlorine-based bleaching. The properties of the T. lanuginosus xylanases are suitable for the high temperatures and alkaline pH's used in bleaching of kraft pulps. Although T. lanuginosus strains and their enzymes are not as thermophilic as those of some eubacteria and archaea that are able to grow near or above 100°C, the organism is relatively easy to cultivate and the enzymes are secreted at much higher levels. Furthermore, the thermostability of xylanases produced by mesophilic organisms is much lower than that of T. lanuginosus. Other secreted enzymes of T. lanuginosus such as α-amylase and phytase also appear to be more thermostable than similar enzymes produced by mesophilic organisms. Enhancement of the thermostability, pH stability and catalytic efficiency of the T. lanuginosus xylanase using molecular manipulation could lead to new applications of this remarkable fungus and its enzymes.
Funding from the ML Sultan Technikon Research Fund and the National Research Foundation (NRF, South Africa) is gratefully acknowledged. The assistance of P. Reddy, P. Biely, Z. Ögel, D. Stephens and K. Rumbold for communication of unpublished results and technical assistance is also acknowledged.