Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-06
2002-08-27
Arthur, Lisa B. (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024330, C536S023200, C435S006120, C435S091100, C435S091200, C435S005000
Reexamination Certificate
active
06441148
ABSTRACT:
FIELD OF THE INVENTION
This application relates to the use of PCR amplification to isolate novel xylanase genes from soil DNA, and to primers useful in such methods and the products obtained thereby.
BACKGROUND OF THE INVENTION
The hydrolysis of cellulose, and hemicellulose, with xylans being a major component of hemicellulose, requires a variety of enzymes having activity as endoglucanases, exoglucanases, and xylanases to work in concert. It is with these systems of enzymes, composed of enzymes from the different cellulase families, that plant material is degraded in nature.
Cellulases have been classified into 12 families (designated A to L), and a single organism may have a set of enzymes with members drawn from several families. Of these families, families F and G show xylanase activity.
There has been an increasing awareness of the potential industrial uses for cellulases and xylanases; examples include biomass conversion, Saddler, J. N.,
Bioconversion of forest and agricultural plant residues,
CAN International, Oxford, England (1993), and the role cellulases and xylanases are playing in pulp processing and paper production. Wick, C. B.,
Genetic Engineering news
14: 10-11 (1994). For example, xylanases can be used to make pulp bleaching more environmentally friendly by reducing organochlorine discharges. McCubbin, N.,
Pulp
&
Paper Canada,
95: 4 (1994).
In identifying and characterizing cellulases and xylanases suitable for use in industry, traditional methods of isolation and selection of cellulase and xylanase-producing organisms continues to be carried out by growth on cellulose and cellulose-like substrates. However, the traditional methods are only suitable for culturable organisms. Considering that it is estimated that only 1% of the organisms present in soil are culturable, Tiedje, J. M.,
ASM News
60:524-525 (1994), these traditional methods only skim the surface of the resource of enzymes which soil could theoretically provide.
Bergquist et al., in a paper delivered at the Society for Industrial Microbiology Meeting in Montreal, Canada in June 1994 discussed methods for isolating hemicellulolytic enzymes from the extremely thermophilic bacteria in hot pools having temperatures as high as 95° C. For non-culturable organisms, they suggest that the polymerase chain reaction (PCR) on total DNA isolated from concentrated hot springs water with primers hybridizing to conserved regions of the known xylanase genes can be used to isolate xylanase DNA. Bergquist did not disclose or suggest methods for recovery of xylanase DNA from far more complex and challenging soil samples.
It is an object of the present invention to provide access to the cellulase and xylanase enzymes produced by non-culturable organisms by providing a mechanism for extracting DNA specific to Family F xylanases from soil.
It is a further object of this invention to provide specific compositions, particularly primers, useful in performing this isolation procedure.
It is still a further object of the invention to provide novel xylanase enzymes containing active sites which have been isolated from soil using the procedures of the present invention.
SUMMARY OF THE INVENTION
The present invention provides a method for recovering xylanase-encoding DNA from soil, comprising the steps of:
(a) treating a soil sample to render DNA in the soil accessible for hybridization with oligonucleotide primers;
(b) combining the treated soil sample with first and second primers in an amplification reaction mixture, said first and second primers hybridizing with conserved regions of the sense and antisense strands respectively of a gene encoding a xylanase and flanking a region of interest in the gene;
(c) thermally cycling the amplification reaction mixture through a plurality of cycles each including at least a denaturation phase and a primer extension phase to produce multiple copies of the region of interest flanked by the primers; and
(d) recovering the copies of the region of interest from the amplification reaction mixture. Because of the complexity of the soil samples, it is likely that the recovered product will include more than one species of polynucleotide. Thus, these recovered copies may, in accordance with the invention, be cloned into a host organism to produce additional copies of each individual species prior to characterization by sequencing.
Recovered DNA which is found to vary from known xylanases can be used in several ways to facilitate production of novel xylanases for industrial application. First, the recovered DNA, or probes corresponding to portions thereof, can be used as a probe to screen soil DNA libraries and recover intact xylanase genes including the unique regions of the recovered DNA. Second, the recovered DNA or polynucleotides corresponding to portions thereof, can be inserted into a known xylanase gene to produce a recombinant xylanase gene with the sequence variations of the recovered DNA.
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O'Neill, “Structure of the gene encoding the exoglucanase of Cellulomonas fimi”, Gene, vol. 44, pp. 325-330, 1986.*
O'Neill, Genbank Accession No. L11080, 1986.*
Millward-Sadler, “Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens . . . ” Biochem J., 312(pt 1), pp. 39-48, Nov. 1995.*
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Scharf, “Cloning with PCR”, PCR Protocols, 1990.*
Barns et al., 1994, “Remarkable Archael Diversity Detected in a Yellowstone National Park Hot Spring Environment”, Proc. Nat'l Acad. Sci. (USA) 91: 1609-1613.
Bergquist et al., 1994, “Hemicellulolytic Enzymes From Extremely Thermophilic Bacteria—Application of Molecular Genetics to Pulp Bleaching”, poster presented at Society for Industrial Microbiology Meeting, Montreal, Canada, Jun. 1994.
Crameri et al., 1996, “Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling”, Nature Biotechnology 14: 315-319.
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Knaebel and Crawford, 1995, “Extraction and Purification of Microbial DNA from Petroleum-Contaminated Soils and Detection of Low Numbers of Toluene, Octane and Pesticide Degraders by Multiplex Polymerase Chain Reaction and Southern Analysis”, Molecular Ecology 4:579-591.
Matsumura and Ellington, 1996, “DNA Shuffling Brightens Prospects for GFP”, Nature Biotechnology 14:366.
McCubbin, 1994, “A Bleaching Revolution is in Progress”, Pulp & Paper Canada 94: 12-16.
Munro et al., 1995, “A Gene Encoding a Thermophilic Alkaline Serine Proteinase from Thermus sp. Strain Rt41A and Its Expression inEscherichia coli”, Microbiology 141:1731-1738.
Patil et al., 1990, “PCR Amplification of anEscherichia coliGene Using Mixed Primers Containing Deoxyadensine at Ambiguous Positions in Degenerate Amino Acid Codons”, Nucl. Acids Res. 18:3080 [erratum appears at Nucleic Acids Res. 19:3184 (1991)].
Porteous et al., 1994, “An Effective Method to Extract DNA from Environmental Samples for Polymerase Chain Reaction Amplification and DNA Fingerprint Analysi
Radomski Christopher C. A.
Seow Kah Tong
Warren R. Antony J.
Yap Wai Ho
Arthur Lisa B.
Brown Karen E.
Fish & Neave
Goldberg Jeanine
Haley Jr. James F.
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