Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-06-22
2001-05-08
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C536S023200, C536S023740
Reexamination Certificate
active
06228630
ABSTRACT:
FIELD OF INVENTION
The present invention relates to an enzyme with xylanase activity, a method of producing the enzyme, an enzyme preparation containing the enzyme, and use of the enzyme for various industrial purposes.
BACKGROUND OF THE INVENTION
Xylan, a major component of plant hemicellulose, is a polymer of D-xylose linked by beta-1,4-xylosidic bonds. Xylan can be degraded to xylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatic hydrolysis of xylan produces free sugars without the by-products formed with acid (e.g. furans).
Enzymes which are capable of degrading xylan and other plant cell wall polysaccharides are important for the food industry, primarily for baking and in fruit and vegetable processing such as fruit juice production or wine making, where their ability to catalyse the degradation of the backbone or side chains of the plant cell wall polysaccharide is utilised (Visser et al., Xylans and.Xylanases, 1991).
Other applications for xylanases are enzymatic breakdown of agricultural wastes for production of alcohol fuels, enzymatic treatment of animal feeds for hydrolysis of pentosans, manufacturing of dissolving pulps yielding cellulose, and bio-bleaching of wood pulp (Detroym R. W. In: Organic Chemicals from Biomass, (CRC Press, Boca Raton, Fla., 1981) 19-41.; Paice, M. G., and L. Jurasek., J. Wood Chem. Technol. 4: 187-198.; Pommier, J. C., J. L. Fuentes, G. Goma., Tappi Journal (1989): 187-191.; Senior, D. J., et al., Biotechnol. Letters 10 (1988):907-912]. WO 92/17573 discloses a substantially pure xylanase derived from the fungal species
H. insolens
and recombinant DNA encoding said xylanase. The xylanase is stated to be useful as a baking agent, a feed additive, and in the preparation of paper and pulp.
WO 92/01793 discloses a xylanase derived from the fungal species
Aspergillus tubigensis.
It is mentioned, but not shown that related xylanases may be derived from other filamentous fungi, examples of which are Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarlum and Trichoderma. The xylanases are stated to be useful in the preparation of bread or animal feed, in breewing and in reducing viscosity or improving filterability of cereal starch.
Shei et al., 1985, and Fournier et al., 1985 describe purification and characterization of endoxylanases isolated from
A. niger.
WO 91/19782 and EP 463 706 discloses xylanase derived from
Aspergillus niger
origin and the recombinant production thereof. The xylanase is stated to be useful for baking, brewing, in the paper making industry, and in the treatment of agricultural waste, etc.
SUMMARY OF THE INVENTION
It is an object of the present invention to prepare single-component xylanases.
Accordingly, the present invention relates to an enzyme exhibiting xylanase activity, which enzyme is immunologically reactive with an antibody raised against a purified xylanase derived from
Aspergillus aculeatus,
CBS 101.43.
In the present context, the term “derived from” is intended not only to indicate a xylanase produced by strain CBS 101.43, but also a xylanase encoded by a DNA sequence isolated from strain CBS 101.43 and produced in a host organism transformed with said DNA sequence.
In another aspect, the invention relates to an enzyme exhibiting xylanase activity, which enzyme is encoded by a DNA sequence comprising at least one of the following partial sequences
(a)
CATCAACATT CATTCATTCA
(SEQ ID No. 7)
(b)
TTTAATTCAT TCCTCAAGCT
(SEQ ID No. 8)
(c)
CAAGAGCAGT CATCCCTTCT
(SEQ ID No. 9)
(d)
TTCCAACATG GTTCAAATCA
(SEQ ID No. 10)
(e)
AAGCAGCTGC TCTGGCTGTC
(SEQ ID No. 11)
(f)
CTTTTCGCCA GCAATGTGCT
(SEQ ID No. 12)
(g)
CTCCAACCCC ATCGAGCCCCG
(SEQ ID No. 13)
(h)
CCAGGCCTCG GTGAGCATCGA
(SEQ ID No. 14)
(i)
TGCCAAATTA CAAGGCGCACG
(SEQ ID No. 15)
(j)
CAAGAAGTAC CTGGGCACCAT
(SEQ ID No. 16)
(k)
GAACCCCCAC AATCACGCAA
(SEQ ID No. 17)
(l)
AAATGGTCGG ACTGCTTTCA
(SEQ ID No. 18)
(m)
ATCACCGCGG CGCTTGCCG
(SEQ ID No. 19)
(n)
CTGTGTTGCC AAACATTGTC
(SEQ ID No. 20)
(o)
TCTGCCGTTG GTCTGGATCA
(SEQ ID No. 21)
(p)
GGCTGCAGTT GCCAAAGGAC
(SEQ ID No. 22)
(q)
TTCAATACTT TGGCACAGCT
(SEQ ID No. 23)
(r)
ACGGATAATC CCGAGCTCAC
(SEQ ID No. 24)
(s)
GGATATTCCAT ACGTTACTCA
(SEQ ID No. 25)
(t)
GCTGAACAAC ACCGCGGACT
(SEQ ID No. 26)
(u)
TTGGTCAAAT TACCCCTGGAAAC
(SEQ ID No. 27)
(v)
TCGATGAAGT GGGATGCCAC
(SEQ ID No. 28)
(w)
AGAACCATCT CAGGGCACCTTC
(SEQ ID No. 29)
(x)
ACGTTCACGA AAGGC
(SEQ ID No. 30)
(y)
CTTCTACTTA GTATTCA
(SEQ ID No. 31)
(z)
CTGACTTACC ATGGCTCGCC
(SEQ ID No. 32)
(A)
TATCTCAGTT CCTTCTGGCC
(SEQ ID No. 33)
(B)
TGCGCTCTTG CAGTCAAAG
(SEQ ID No. 34)
(C)
CCTTCGCTGC CCCCGCCGCC
(SEQ ID No. 35)
(D)
GAGCCCGTCG AGGAACGGGG
(SEQ ID No. 36)
(E)
CCCTAACTTC TTTTCTGCCC
(SEQ ID No. 37)
(F)
TTGCTGGGCG CTCGACTGG
(SEQ ID No. 38)
(G)
CAGCTCCACT GGCTACTCGAA
(SEQ ID No. 39)
In further aspects the invention relates to an enzyme exhibiting xylanase activity, which enzyme is encoded by a DNA sequence comprised in or comprising a DNA sequence shown in any of SEQ ID Nos. 1, 3 or 5, respectively, or sequence homologous thereto encoding a polypeptide with xylanase activity.
The enzyme encoded by the DNA sequence shown in SEQ ID No. 1 is termed xylanase I (or xyl I) in the following disclosure, the enzyme encoded by the DNA sequence SEQ ID No. 3 is termed xylanase II (or xyl II) in the following disclosure, and the enzyme encoded by the DNA sequence SEQ ID No. 5 is termed xylanase III (or xyl III) in the following disclosure.
In a further aspect, the invention relates to an enzyme exhibiting xylanase activity, which enzyme is encoded by a DNA sequence comprising the following partial sequence
(SEQ ID No. 40)
CATCAACATT CATTCATTCA TTTAATTCAT TCCTCAAGCT
CAAGAGCAGT CATCCCTTCT TTCCAACATG GTTCAAATCA
AAGCAGCTGC TCTGGCTGTC CTTTTCGCCA GCAATGTGCT
CTCCAACCCC ATCGAGCCCC GCCAGGCCTC GGTGAGCATC
GATGCCAAAT TCAAGGCGCA CGGCAAGAAG TACCTGGGCA CCAT
or a sequence homologous thereto encoding a polypeptide with xylanase activity. A particular example of such enzyme is xylanase I as defined above.
In a still further aspect, the invention relates to an enzyme exhibiting xylanase activity, which enzyme is encoded by a DNA sequence comprising the following partial sequence
(SEQ ID NO. 41)
AAAATGGTCG GACTGCTTTC AATCACCGCG GCGCTTGCCG
CGACTGTGTT GCCAAACATT GTCTCTGCCG TTGGTCTGGA
TCAGGCTGCA GTTGCCAAAG GACTTCAATA CTTTGGCACA
GCTACGGATA ATCCCGAGCT CACGGATATT CCATACGTTA
CTCAGCTGAA CAACACCGCG GACTTTGGTC AAATTACCCC
TGGAAACTCG ATGAAGTGGG ATGCCACAGA ACCATCTCAG
GGCACCTTCA CGTTCACGAAAGGCG
or a sequence homologous thereto encoding a polypeptide with xylanase activity. A particular example of such enzyme is xylanase II as defined above.
In a still further aspect, the invention relates to an enzyme exhibiting xylanase activity, which enzyme is encoded by a DNA sequence comprising the following partial sequence
(SEQ ID No. 42)
TCCCTTCTAC TTAGTATTCA CTGACTTACC ATGGCTCGCC
TATCTCAGTT CCTTCTGGCC TGCGCTCTTG CAGTCAAAGC
CTTCGCTGCC CCCGCCGCCG AGCCCGTCGA GGAACGGGG
CCTAACTTCT TTTCTGCCCT TGCTGGGCGC TCGACTGGCA
GCTCCACTGG CTACTCGAA
or a sequence homologous thereto encoding a polypeptide with xylanase activity. A particular example of such enzyme is xylanase III as defined above.
In the present context, the term “homologue” is intended to indicate a polypeptide encoded by DNA which hybridizes to the same probe as; the DNA coding for the xylanase enzyme under certain specified conditions (such as presoaking in SXSSC and prehybridizing for 1 h at ~40° C. in a solution of 5×SSC, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 &mgr;g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 &mgr;Ci 32-P-dCTP labelled probe for 18 h at ~40° C. followed by washing three times in 2×SSC, 0.2% SDS at 40° C. for 30 minutes). More specifically, the term is intended to refer to a DNA sequence which is at least 70% homologous to any of the sequences shown above encoding a xylanase of the invention, such as at least 75%, at least 80%, at least 85%,
Andersen Lene Nonboe
Christgau Stephan
Dalbøge Henrik
Heldt-Hansen Hans Peter
Jacobsen Tina Sejersgåard
Lambiris Esq. Elias J.
Nashed Nashaat T.
Novo Nordisk A S
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