Invented by Ryszard Brzezinski, Claude V. Dery, Carole Beaulieu, AB Enzymes GmbH
The AB Enzymes GmbH invention works as followsThe present invention is a method for chemically treating biomass of plants with an enzyme system which retains its function even at high temperatures and low pH. The present invention describes enzyme preparations that are enriched with xylanase, which can retain their activity at low pH and high temperatures. These preparations can be used in their crude, unpurified state and are particularly useful in the manufacture of pulp and papers.
Background for Thermostable xylanase protein, DNA and methods of Use
Xylan is a polymer that is a major component in hemicellulose. It consists of a backbone made of? 1,4-linked Dxylose residues with glucuronic and?-Larabinofuranose side chains. (Timell, T. E. et al. Wood Sci. Technol. 1:45-70 (1967)). Xylan, after cellulose is the second-most abundant carbohydrate fraction in plant biomass. Xylan is gaining attention recently as a renewable resource.
It is complex and requires more than one enzyme to degrade xylan into soluble monomers. Many hemicellulases can hydrolyze xylan, including?-1. 4-xylanases(EC 18.104.22.168),?-xylosidases, and several debranching enzymatics (Biely P. Trends Biotechnol 3:1286-290 (1985); Dekker R. F. H. in Hignehi T. (ed. ), Biosynthesis and Biodegradation Of Wood Components (Academic Woodward, J. Top Enzyme Ferment. Biotechnol. 8:9-30 (1984)). These enzymes are important in decomposing soil plant litter. They have been studied extensively in both bacteria and fungi. Rev. Rev. 139:187-194 (1993)).
The xylanases found in both prokaryotes as well as eukaryotes are secreted by a variety of microorganisms. Carbohydrate Chem. Biochem. 32:277-352 (1976)). Multiple xylanases are produced by xylanolytic bacteria to attack different bonds within these molecules. The xylanases that have been characterized so far fall into two categories: the high Mr/low pI and the low Mr/high pI classes, which correspond to the glycosyl hydrolase families 10 and 11. (Henrissat & Bairoch Biochem). J. 293:781-788 (1993)).
The cloning xylanases from Actinomadura Sp. has been reported. FC7 (Ethier, J. -F. et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics, R. Baltz et al., eds, (Proc. 5th ASM Conference. Gen. Mol. Biol. Indust. Microorg., October 11-15, 1992 in 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. Microbiol. 16:9874 (1988); Ito, K. et al., Biosci. Biotec. Biochem. Biochem. 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 are tightly bound with cellulose and lignin in plant biomass.” In the pulp-and-paper industry, during the chemical pulping of wood (cooking), the majority of lignin must be removed to obtain a cellulose pulp. The resulting pulp, however, is brown due to the small amount of lignin that remains in the pulp. This residual lignin has traditionally been removed using a multi-stage procedure that uses a typical combination of chlorine chemicals and extraction stages. When the use of chlorine chemicals should be minimized or completely avoided, peroxide and oxygen are used.
The use of enzymes in the bleaching of kraft paper can reduce the amount of chemicals used for subsequent bleaching, or increase the brightness of the pulp. (Kantelinen, Viikari, Paper and Timber, 7:384-389, 1991; and Kantelinen, “Enzymes in Bleaching of Kraft Pulp,” Dissertation, Technical Research Centre of Finland VTT Publications No. 114, Espoo 1992). In this case, hemicellulose must be free from cellulases that could damage the cellulose fibers.
The use of hemicellulose-hydrolyzing enzymes for different bleaching sequences are discussed in WO89/08738, WO91/02791, WO91/05908, EP 383,999 and WO91/05908
Hemicellulolytics enzymes can be used to improve drainage of recycled pulp or hemicellulolytics enzymes can be used in the production dissolving pulps (Viikari et al., “Hemicellulases for Industrial Applications, ” In: Bioconversion of Forest and Agricultural Wastes. Saddler J., ed. CAB International USA (1993)). Hemicellulolytics enzymes are used to improve the drainage of recycled paper or in the production dissolving pulps.
The use of hemicellulolytic enzymatic enzymes to improve water removal from mechanical paper is discussed in the EP 262,040 and EP 334 739, and EP 351,655 as well as DE 4,000,558). It is important to convert both cellulose as well as hemicellulose when hydrolyzing biomass into liquid fuels or chemical products (Viikari, et. al. “Hemicellulases For Industrial Applications”, In: Bioconversion Of Forest And Agricultural Wastes. Saddler, J. ed. CAB International, USA, 1993). In the feed industry there is also a need for a combination of enzymes that can degrade high?-glucans and hemicellulose substrates.
To be amenable to enzymatic hydrolysis in vitro, the cellulose-hemicellulose-lignin matrix must be chemically pretreated. Thermo-mechanical steam treatment is followed by extraction using hot water. Microbiol. 1:355-361 (1987)). “A mildly acidic liquid is obtained which contains some water-soluble hemicellulose chain and lignin derivates.
There is therefore a need for xylanases that retain their activity in industrial ambient conditions.” In the paper industry, xylanase is needed to function in high-temperature, acidic liquids produced by thermomechanical steam treatment or hot water extraction.
The inventors searched for a microbe that could be a source of enzymes. They were aware of the importance of finding an environmentally friendly and economic method to chemically modify plant biomass in order to enzymatically treat it under harsh conditions such as high temperatures and low pH.
These studies led to the isolation and identification a novel thermophilic strain of actinomycete Actinomadura. FC7. Actinomadura sp. “FC7 expresses XYL 1 and XYL 2 which retain a high amount of enzymatic activities at low pH and high temperatures.
The invention also relates to DNA encoding XYLI or XYLII, as well as recombinant host cells transformed with this DNA.
The invention also relates to purified XYL I or XYL II and enzyme preparations containing XYL I or XYL II.
The invention also relates to a biobleaching method, which uses the enzyme preparations.
In the following description, several terms related to recombinant-DNA technology are used extensively. The following definitions will help to clarify the claims and specifications, and the extent to which they apply.
Xylanase. A xylanase, as used herein is a hemicellulase which cuts the?-1.4 bonds in the xylosic chains of xylan (xylan being a polymer made up of D-xylose residues linked by?-1.4 linkages. “Xylanase is synonymous with xylanolytic activities.
By a host which is “substantially incapable” of synthesizing a cellulase, it is meant that the activity of a cellulase is reduced, deficient or absent in comparison to the wild type.
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