Xylan, a major component of hemicellulose, is a polymer consisting of a backbone of .beta.(1,4)-linked D-xylose residues (often acetylated) with .alpha.-L-arabinofuranose and glucuronic acid side chains (Timell, T. E., et al., Wood Sci. Technol. 1:45-70 (1967)). After cellulose, xylan is the second most abundant carbohydrate fraction of plant biomass. Xylan has recently received increased attention as a renewable bioresource.
Being complex, more than one enzyme is required to completely degrade xylan to soluble monomers. Xylan can be hydrolyzed by many hemicellulases, such as, for example, .beta.-1,4-xylanases (EC 3.2.1.8), .beta.-xylosidases and several debranching enzymes (Biely, P., Trends Biotechnol 3:286-290 (1985); Dekker, R. F. H., in Hignehi, T., ed., Biosynthesis and biodegradation of wood components (Academic Press Inc., Orlando), pp. 505-533 (1985); Woodward, J., Top Enzyme Ferment. Biotechnol. 8:9-30 (1984)). The activities of these enzymes play an important role in the decomposition of soil plant litter and have been extensively studied both in bacteria and fungi (Wong, K. K. Y. et al., Microbiol. Rev. 52:305-317 (1988); Poutanen, K. et al., in Enzymes in biomass conversion (ACS Symposium series 460), Leatham & Himmel, eds., American Chemical Society, Washington, D.C. (1991), pp.426-436; Gilbert & Hazlewood, J. Gen. Microbiol. 139:187-194 (1993)).
Various microorganisms secrete enzymes that are capable of degrading xylans, and xylanases have been found in both prokaryotes and eukaryotes (Dekker, R. F. H., Richards, G. N., Adv. Carbohydrate Chem. Biochem. 32:277-352 (1976)). Xylanolytic micro-organisms often produce multiple xylanases to attack the different bonds in these molecules. All the xylanases so far characterized fall into two classes: the high M.sub.r /low pI class and the low M.sub.r /high pI class which coincide, respectively, with the families 10 and 11 of glycosyl hydrolases (Henrissat & Bairoch, Biochem. J. 293:781-788 (1993)).
The cloning of xylanases has been reported from Actinomadura sp. FC7 (Ethier, J. -F. et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, R. Baltz et al., eds, (Proc. 5th ASM Conf. Gen. Mol. Biol. Indust. Microorg., Oct. 11-15, 1992, Bloomington, Ind., poster C25); bacteria (e.g. Ghangas, G. S. et al., J. Bacteriol. 171:2963-2969 (1989); Lin, L. -L., Thomson, J. A., Mol. Gen. Genet. 228:55-61 (1991); Shareck, F. et al., Gene 107:75-82 (1991); Scheirlinck, T. et al., Appl Microbiol Biotechnol. 33:534-541 (1990); Whitehead, T. R., Lee, D. A., Curr. Microbiol. 23:15-19 (1991)); and fungi (Boucher, F. et al., Nucleic Acids Res. 16:9874 (1988); Ito, K. et al., Biosci. Biotec. Biochem. 56:906-912 (1992); Maat, J. et al., in Visser, J. et al., eds., Xylans and Xylanases (Elsevier Science, Amsterdam), pp. 349-360 (1992); van den Broeck, H. et al., EP 463,706 A1 (1992), WO 93/25671 and WO 93/25693).
The xylan-containing hemicelluloses in plant biomass are tightly bound to cellulose and lignin. In the pulp and paper industry, in chemical pulping (cooking) of the wood, the major part of the lignin is extracted to get acceptable cellulose pulp product. However, the resulting pulp is brown, mainly because of the small portion of the lignin still remaining in the pulp after cooking. This residual lignin is traditionally removed in a multi-stage bleaching procedure using typically a combination of chlorine chemicals and extraction stages. Peroxide, oxygen and ozone are also used when the use of the chlorine chemicals is wanted to be reduced or avoided totally.
Hemicellulases can be used in enzyme-aided bleaching of pulps to decrease chemical dosage in subsequent bleaching or to increase brightness of the pulp (Kantelinen et al., International Pulp Bleaching Conference, Tappi Proceedings, 1-5 (1988); Viikari et al., Paper and Timber 7:384-389 (1991); and Kantelinen et al., "Enzymes in bleaching of kraft pulp," Dissertation for the degree of Doctor of Technology, Technical Research Centre of Finland, VTT Publications 114, Espoo, 1992). Naturally, in this use, the hemicellulose should be free of cellulases, which would harm the cellulose fibers.
The use of hemicellulose hydrolyzing enzymes in different bleaching sequences is discussed in WO 89/08738, EP 383,999, WO 91/02791, EP 395,792, EP 386,888, EP 473,545, EP 489,104 and WO 91/05908.
Other industrial applications for hemicellulolytic enzymes are in the production of thermo-mechanical pulps, where the aim of the use of hemicellulolytic enzymes is decreased energy consumption. Hemicellulolytic enzymes can be used to improve drainage of recycled pulp or hemicellulolytic enzymes can be used in the production of dissolving pulps (Viikari et al., "Hemicellulases for Industrial Applications, " In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)).
The use of hemicellulolytic enzymes for improved water removal from mechanical pulp is discussed in EP 262,040, EP 334,739 and EP 351,655 and DE 4,000,558). When the hydrolysis of biomass to liquid fuels or chemicals is considered, the conversion of both cellulose and hemicellulose is essential to obtain a high yield (Viikari et al., "Hemicellulases for Industrial Applications," In: Bioconversion of Forest and Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)). Also, in the feed industry, there is a need to use a suitable combination of enzyme activities to degrade the high .beta.-glucan and hemicellulose containing substrate.
To be amenable to enzymatic hydrolysis in vitro, the cellulose-hemicellulose-lignin matrix must be chemically pretreated. One of such procedures involves a thermo-mechanical steam treatment followed by extraction with hot water (Chahal, D. S. et al., J. Indust. Microbiol. 1:355-361 (1987)). A mildly acidic liquor is obtained, which contains water-soluble hemicellulose chains and some lignin derivatives.
However, to ensure further enzymatic hydrolysis of the xylan chains into oligomers or monomers, enzyme systems that are efficient at conditions combining high temperature (such as 70.degree. C.) and moderately acidic pH (around 4.0) are needed. The combination of these two parameters seems however to be harmful for the majority of known xylanases. For instance, at pH 4, xylanase II from the mesophilic actinomycete Streptomyces roseiscleroticus (a low M.sub.r /high pI enzyme) retains less than 5% of the activity it had at pH 6.0-6.5 (Grabski & Jeffries, Appl. Environ. Microbiol. 57:987-992 (1991)). The crude xylanase from Aureobasidium pullulans (Myburgh, J. et al., Proc. Biochem. 26:343-348 (1991)) is acidophilic, having a pH optimum between 3.5 and 4.0 but its activity sharply decreases at temperatures higher than 35.degree. C. The thermostable xylanase from the fungus Thermoascus aurantiacus retains at pH 3.5, only 12% of its maximal activity (Tan L. U. L. et al., Can. J. Microbiol. 33:689-692 (1987)). Another xylanase, a high M.sub.r /low pI enzyme from the extremophile bacterium "Caldocellum saccharolyticum" was shown to be very stable at 60.degree. C. but retained little activity below pH 5 (Luthi, E. et al., Appl. Environ. Microbiol. 56:2677-2683 (1990); Luthi, E. et al., Appl. Environ. Microbiol. 56:1017-1024 (1990). Crude xylanases from various Actinomadura isolates were stable for many hours when incubated at 70.degree.-75.degree. C., but retained less than 15% of their activity at pH 4.0-4.5 and 70.degree. C. (Holtz, C. et al., Antonie van Leeuwenhoek 59:1-7 (1991)).
Thus, there is a need for enzyme preparations that contain xylanases which retain activity under industrial ambient conditions. Especially in the paper manufacturing industry, there is a need for xylanase preparations that are functional in the high temperature, acidic liquor produced by thermo-mechanical steam treatment and hot water extraction.