Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1997-11-19
2001-04-10
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S252300, C435S254300, C435S277000, C435S320100, C536S023200, C536S024300
Reexamination Certificate
active
06214598
ABSTRACT:
FIELD OF INVENTION
The present invention relates to enzymes with endoglucanase activity, a method of producing the enzymes, and an enzyme preparation containing an enzyme of the invention.
BACKGROUND OF THE INVENTION
Endoglucanases (EC no. 3.2.1.4) constitute a group of hydrolases, which catalyse endo hydrolysis of 1,4-&bgr;-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose) lichenin, &bgr;-1,4 bonds in mixed &bgr;-1,3 glucans such as cereal &bgr;-D-glucans or xyloglucans and other plant material containing cellulosic parts. The authorized name is endo-1,4-&bgr;-D-glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. Reference can be made to R. F. Gould, “Cellulases and their Application”, Advances in Chemistry Series 55, American Chemical Society (1969), T. M. Wood, “Properties and Mode of Action of Cellulases”, in Biotechnology and Bioengineering Symposium, no. 5, John Wiley, 111-137 (1975), Y. -H. Lee and L. T. Fan, “Properties and Mode of Action of Cellulose”, Advances in Biochemical engineering 17, 101-129 (1980), J. Goksyr and J. Eriksen, “Cellulases” in A. H. Rouse, Microbial Enzymes and Bioconversions, Academic Press, 283-330 (1980), T. -M. Enveri, “Microbial Cellulases” in W. M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, 183-224 (1983).
Endoglucanases have been found to be produced by various types of organisms such as plants and microorganisms, and endoglucanases of a wide variety of specificities have been identified. For instance, xyloglucan specific endoglucanases have been identified in various plants, cf the disclosure of Fry et al. (1992), Nishitani and Tominaga (1992), Hayashi et al. (1984), McDougall and Fry (1991), and WO 93/17101. All of these enzymes have been found to have transferase activity (as defined e.g. by Fry et al., 1992 and Nishitani et al., 1992) and are not, accordingly, classified as a real endoglucanase. Hitherto, xyloglucan specific endoglucanases have not been identified in microorganisms.
Microbial endoglucanases have been described by Beldman et al., 1985 (
Trichoderma viride
) and in WO 93/20193 (
Aspergillus aculeatus
), the latter reference being published only after the priority dates of the present invention. Furthermore, Sharma et al., 1991, Ooi, et al., 1990, and Gilkes et al. 1991 describe microbial endoglucanases.
Endoglucanases may advantageously be used for the degradation of cellulose components present in plant cell walls and bacterial polysaccharides. Endoglucanases having a high xyloglucan-degrading activity may be of particular use for degradations of cell wall material having a high xyloglucan content, for instance in the wine and fruit industry, for pectin-extraction and for removal of hemicelluloses from textile fibres.
The object of the invention is to provide novel endoglucanases exhibiting useful substrate specificities and a method for producing the endoglucanases in a better yield and higher purity than hitherto possible. A further object is to provide novel products, wherein the proportion of the endo-&bgr;-1,4-glucanase is either increased or decreased relative to the proportion in the original product. The endoglucanases and the novel products of the invention, whether alone or in combination with other enzymes may be used for the degradation of plant cell wall tissue.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect the present invention relates to an enzyme exhibiting endoglucanase activity, which enzyme is encoded by a DNA sequence comprising at least one of the following partial sequences
(a) ATTCATTTGTG GACAGTGGAC (SEQ ID No: 1)
(b) GTTGATCGCA CATTGAACCA (SEQ ID No: 2)
(c) ACCCCAGCCG ACCGATTGTC (SEQ ID No: 3)
(d) CTTCCTTACC TCACCATCAT (SEQ ID No: 4)
(e) TTAACATCTT TTCACCATGA (SEQ ID No: 5)
(f) AGCTTTCCCT TCTCTCCCTT (SEQ ID No: 6)
(g) GCCACCCTGG CTTCCGCTGC CAGCCTCC (SEQ ID No: 7)
(h) GACAGTAGCA ATCCAGCATT (SEQ ID No: 8)
(i) AGCATCAGCC GCTTTGTACA (SEQ ID No: 9)
(j) CCATGAAGTT CACCGTATTG (SEQ ID No: 10)
(k) GCACTGCTTC TCTCCCAGGT (SEQ ID No: 11)
(l) GTGGGCGGCC CCTCAGGCAA (SEQ ID No: 12)
(m) ACGCTCCTCC AATTTTCTCT (SEQ ID No: 13)
(n) GGCTTGGTAG TAATGAGTCT (SEQ ID No: 14)
(o) GGCGCAGAGT TTGGCCAGGC (SEQ ID No: 15)
(p) CAACATCCCC GGTGTTCTGGG (SEQ ID No: 16)
In the present context, the term “endoglucanase activity” is intended to indicate a capability of hydrolysing 1,4-&bgr;-D-glycosidic linkages present in any cellulosic material, such as cellulose, cellulose derivatives, lichenin, &bgr;-D-glucan, or xyloglucan. The endoglucanase activity may be determined in accordance with methods known in the art, examples of which are described in the Materials and Methods and Examples section herein. One unit of endoglucanase activity (e.g. CMCU, AVIU, XGU or BGU) is defined as the production of 1 &mgr;mol reducing sugar/min from a glucan substrate, the glucan substrate being, e.g., CMC (CMCU), acid swollen Avicell (AVIU), xyloglucan (XGU) or cereal &bgr;-glucan (BGU). The reducing sugars are determined as described in the Materials and Methods section herein. The specific activity of an endoglucanase towards a substrate is defined as units/mg of protein.
In a further aspect, the invention relates to an enzyme exhibiting endoglucanase activity, which enzyme
i) is encoded by a DNA sequence comprising or included in at least one of the following partial sequences
AAAGATTCATTTGTGGACAGTGGACGTTGATCGCACATTGAACCAACCCCAGCCGACCGATTGTCCTTCCTTACCTCACCATCATTTAACATCTTTTCACCATGAAGCTTTCCCTTCTCTCCC TTGCCACCCTGGCTTCCGCTGCCAGCCTCCAGCGCCGCACACTTCTGCGGTCAGTGGGATA CCGCCACCGCCGGTGACTTCACCCTGTACAACGACCTTTGGGGCGAGACGGCCGGCACCGG CTCCCAGTGCACTGGAGTCGACTCCTACAGCGGCGACACCATCGCTTGTCACACCAGCAGG TCCTGGTCGGAGTAGCAGCAGCGTCAAGAGCTATGCCAACG (SEQ ID No: 17) or
CAGCATCTCCATTGAGTAATCACGTTGGTGTTCGGTGGCCCGCCGTGTTGCGTGGCGGAGG CTGCCGGGAGACGGGTGGGGATGGTGGTGGGAGAGAATGTAGGGCGCCGTGTTTCAGTCCC TAGGCAGGATACCGGAAAACCGTGTGGTAGGAGGTTTATAGGTTTCCAGGAGACGCTGTAT AGGGGATAAATGAGATTGAATGGTGGCCACACTCAAACCAACCAGGTCCTGTACATACAAT GCATATACCAATTATACCTACCAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID No: 18)
or a sequence homologous thereto encoding a polypeptide with endoglucanase activity,
ii) is immunologically reactive with an antibody raised against a highly purified endoglucanase encoded by the DNA sequence defined in i) and derived from
Aspergillus aculeatus
, CBS 101.43, and/or
iii) is specific for xyloglucan.
In the present context, the term “specific for xyloglucan” is intended to indicate that the enzyme exhibits a high activity on a xyloglucan substrate and only low, if any, activity on other cellulose-containing substrates such as carboxymethyl cellulose, cellulose, or other glucans.
The specificity of an endoglucanase towards xyloglucan may be defined as a relative activity determined as the release of reducing sugars at optimal conditions obtained by incubation of the enzyme with xyloglucan and the other substrate to be tested, respectively. For instance, the specificity may be defined as the xyloglucan to &bgr;-glucan activity (XGU/BGU) or xyloglucan to carboxy methyl cellulose activity (XGU/CMCU) or xyloglucan to acid swollen Avicell activity (XGU/AVIU).
In the following disclosure, the enzyme defined by properties i)-iii) above is referred to as of endoglucanase type II or (for short Endoglucanase II or EG II).
In the present context, the term “derived from” is intended not only to indicate an endoglucanase produced by strain CBS 101.43, but also an endoglucanase encoded by a DNA sequence isolated from strain CBS 101.43 and produced in a host organism transformed with said DNA sequence.
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 an endoglucanase enzyme of the invention under certain specified conditions (such as presoaking in 5×SSC and prehybridizing for 1 h at ~40° C. in a solution of 5×SSC, 5×Denhardt's solution, and 50 &mgr;g of denatured sonicated calf thymus DNA, followed by hybrid
Andersen Lene Nonboe
Christgau Stephan
Dalboege Henrik
Kauppinen Markus Sakari
Kofod Lene Venke
Achutamurthy Ponnathapu
Gregg Valeta
Moore William W.
Novo Nordisk A S
Zelson Steve T.
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