Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-05-12
2004-01-27
Rao, Manjunath (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S200000, C435S069100, C435S252300, C435S320100, C536S023200, C510S114000, C510S392000, C510S515000
Reexamination Certificate
active
06682923
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to xylanase activity (XA) proteins and nucleic acids. The invention further relates to the use of the XA proteins in the bleaching process of pulp and in the food and animal feed industry.
BACKGROUND OF THE INVENTION
Glycosyl hydrolase enzymes have been classified into more than 60 families that include xylanases (Xyn), cellulases, mannanases, amylases, beta-glucanases, and other carbohydrases [Henrissat, Biochem. J. 280:309-316 (1991); Henrissat and Bairoch, Biochem. J. 293:781-788 (1993); Henrissat and Bairoch, Biochem. J. 316:695-696 (1996); Davies and Henrissat, Structure 3:853-859 (1995); Coutinho and Henrissat, in Genetics, Biochemistry and Ecology of Cellulose Degradation, eds. Ohmiya et al., Uni Publishers Co., Tokyo, pp 15-23 (1999)]. These enzymes are classified based on amino acid sequence, the three dimensional structure and the geometry of the catalytic site [Gilkes et al., Microbiol. Reviews 55:303-315 (1991)]. Xylanases are produced by many organisms of bacterial and fungal origin (
Enzymes for Pulp and Paper Processing
, eds. Jeffries and Viikari; ACS Symposium Series Vol.655, American Chemical Society, Washington, D.C. (1996); and Xylans and Xylanases, eds. Visser et al., Progress in Biotechnology Vol. 7; Amsterdam-London-New York-Tokyo (1992)] and are used to hydrolyze the polysaccharide xylan, which is a major component of the plant cell walls [
Hemicellulose and Hemicellulases
, eds. Coughlan & Hazelwood; Portland Press Ltd, London-Chapel Hill, (1993)].
The endo-beta-1,4-xylanases (EC 3.2.1.8) belong either to the family 10 xylanases, formerly known as F, or to the family 11 xylanases, also known as G. The family 10 have an (&agr;/&bgr;)
8
barrel fold [Dominguez et al., Nat. Struc. Biol. 2:29-35 (1995)], whereas the family 11 xylanases are mostly &bgr;-sheet and the overall structure resembles that of a right hand [Torronen et al., EMBO J. 13:2493-2501 (1994)]. The
Bacillus circulans
xylanase belongs to the family 11.
Family 11 xylanases have been reported from varies microorganism (bacteria, yeast and fungi), including
Aspergillus awamori
var.
kawachi xyn
A [Ito, Swiss prot. Entry P48824
]; Aspergillus niger
Xyn A [Krengel and Dijkstra, J. Mol. Biol. 263(1):70-78 (1996); PDB entry 1 ukr];
Aspergillus kawachii
Xyn C [Ito et al., Biosci. Biotechnol. Biochem. 56(8):1338-1340 (1992)];
Aspergillus tubigensis
Xyn A [de Graaff et al., Mol. Microbiol. 12(3):479-490 (1994)];
Bacillus circulans
Xyn A [Yang et al., Nucl. Acids. Res., 16:7187 (1988)];
Bacillus pumilus
Xyn A [Fukusaki et al., FEBS Lett. 171:197-201 (1984);
Bacillus subtilis
Xyn A [Paice et al., Arch, Microbiol. 144:201-202 (1986)]; Bacillus sp. strain 41M-1 [Ryuichiro et al., Nucl. Acids Symp. Series 31:235-236 (1994)];
Cellulomonas fimi
Xyn D; Chainia spp. Xyn;
Clostridium acetobutylicum
Xyn B [Zappe et al., Nucl. Acids Res. 18(8):2179 (1990);
Clostridium stercorarium
; Xyn A [Sakka et al., Biosci. Biotechnol. Biochem. 57(2):273-277 (1993)];
Cochliobolus carbonum
[Apel et al., Mol. Plant Microbe Interact. 6(4):467-473 (1993)];
Fibrobacter succinogenes
Xyn C [Paradis et al., J. Bacteriol. 175(23):7666-7672 (1993)];
Neocallimastix patriciarum
Xyn A [Gilbert et al., Mol. Microbiol. 6(15):2065-2072 (1992)];
Nocardiopsis dassonvillei
Xyn II;
Paecilomyces varotii
[J. Mol. Biol. 243(4):806-808 (1994); PDB entry 1 PVX];
Ruminococcus flavefaciens
Xyn A Zhang and Flint, Mol. Microbiol. 6(8):1013-1023 (1992)];
Schizophyllum commune
Xyn [Yaguchi et al., in Xylans and Xylanases, eds. Visser et al., Progress in Biotechnology Vol. 7; pp 149-154, Amsterdam-London-New York-Tokyo (1992);
Streptomyces lividans
Xyn B [Shareck et al., Gene 107(1):75-82 (1991)];
Streptomyces lividans
Xyn C [Shareck et al., Gene 107(1):75-82 (1991)]; Streptomyces sp. No. 36a Xyn [Nagashima et al., Trends Actinomycetoligia 91-96 (1989)];
Streptomyces thermoviolaceus
Xyn II;
Thermomonospora fusca
Xyn A;
Thermomyces lanuginosus
[Gruber et al., Biochemistry 37(39):13475-13485 (1998)];
Trichoderma harzianum
Xyn [Campbell et al., PDB entry 1XND];
Trichoderma reesei
Xyn I [Torronen and Rouvinen, Biochemistry 34:847 (1995); PDB entry 1XYN];
Trichoderma reesei
Xyn II [Torronen et al., EMBO J. 13(11):2493-2501 (1994); PDB entry 1 ENX];
Trichoderma viride
Xyn [Yaguchi, GenBank accession #A44594; (gi:627019)].
In recent years, xylanases have become more and more used in the pulp and paper industry in a process called kraft pulp bleaching [
Enzymes for Pulp and Paper Processing
, eds. Jeffries and Viikari; ACS Symposium Series Vol. 655, American Chemical Society, Washington, D.C. (1996)]. These enzymes are added to the pulp before the pulp is bleached, to enhance the bleaching process and to remove a portion of the xylan in the pulp [Paice and Jurasek, J. Wood Chem. Tech. 4(2):187-198 (1984)]. This enzymatic pre-treatment allows the subsequent bleaching chemicals, including chlorine, chlorine dioxide, hydrogen peroxide, oxygen, ozone, and sodium hydroxide, to bleach the pulp more efficiently than in the absence of xylanase treatment. The enhanced efficiency of bleaching has allowed mills to reduce the amount of chlorine-based chemicals used, thereby decreasing the amount of toxic by-products, which are environmental pollutants. In addition, less bleaching chemicals are used, lowering the chemical costs.
The Family 11 xylanases have several advantages over other xylanases in pulp bleaching applications. Most of the Family 11 xylanases are smaller than xylanases in other families. The small size relative to other xylanases is probably beneficial in penetrating the pulp fibers to release xylan from the pulp and enhance the bleaching. The Family 11 xylanases are also “pure” xylanases in terms of their catalytic activity. Unlike the xylanase enzymes in other families, these enzymes hydrolyze only xylan and do not hydrolyze cellulose. Cellulose hydrolysis damages the pulp and is unacceptable in a commercial mill.
In spite of the advantages of Family 11 xylanases in pulp bleaching, these enzymes have significant drawbacks. The range of temperature and pH that the enzymes exhibits activity on pulp are 45° C. to 55° C. and pH 5.0 to 7.5. A small proportion of mills have operated historically within these ranges. However, the step in the process where xylanase is applied is after a hot alkali treatment, so that the pulp is very basic and hot, typically having a temperature of 60° C. to 70° C. and a pH of 10 to 12. Both of these conditions are sub-optimal for xylanase enzymatic activity. For example, the
Bacillus circulans
wild type xylanase has a temperature optimum of 55° C. and a pH optimum of 5.5. In some mills the adjustment of temperature and pH are acceptable and routine, albeit energy intensive and costly. In many mills achieving the desired treatment conditions causes severe problems. Therefore, the intrinsic properties of the enzyme, such as thermostability and activity at elevated pH are critical parameters for their use in the bio-bleaching processes.
Among naturally occurring xylanases, thermostable enzymes have been isolated from thermophilic microbes, such as
Caldocellum saccharolyticum, Thermatoga maritima
and Thermatoga sp. strain FjSS3-B.1, all of which grow at 80° C. to 100° C. [(Luthi et al. Appl. Environ. Microbiol. 56:2677-2683 (1990); Winterhalter and Liebl, Appl. Environ. Microbiol. 61:1810-1815 (1995); Simpson et al., Biochem. J. 277:413-417 (1991)]. However, all are relatively large in size with high molecular mass of 35-120 kDa (320-1100 residues) and as such, their penetration into the pulp fibers might be limited. Some of these xylanases (e.g.,
C. saccharolyticum
xylanase A) belong to families other than Family 11, and have bot
Bentzien Jörg
Dahiyat Bassil I.
Dorsey & Whitney LLP
Kosslak Renee M.
Rao Manjunath
Silva Robin M.
Xencor
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