Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-06-22
2001-02-20
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S263000, C510S392000
Reexamination Certificate
active
06190899
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Technical Field
The present invention relates to cellulase compositions produced by Actinomycete, methods of producing such cellulases and the use of such cellulases. The present invention further relates to the use of the novel cellulase in compositions recognized in the art as advantageously having cellulase added thereto, including, as an additive in a detergent composition, in the treatment of textiles such as cellulose containing fabrics and fibers useful therefor, as an animal feed additive, as a processing aid in baking, in the treatment of pulp and paper and in the treatment of starch for the production of high fructose corn-syrup or ethanol.
B. State of the Art
Cellulases are enzymes which are capable of the hydrolysis of the &bgr;-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and &bgr;-glucosidases (Knowles, J. et al., (1987),
TIBTECH
5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Primary among the applications that have been developed for the use of cellulolytic enzymes are those involving degrading (wood) cellulose pulp into sugars for (bio)ethanol production, textile treatments like ‘stone washing’ and ‘biopolishing’, and in detergent compositions. Thus, cellulases are known to be useful in detergent compositions for removing dirt, i.e., cleaning. For example, Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826 illustrate improved cleaning performance when detergents incorporate cellulase. Additionally, Great Britain Application No. 1,358,599 illustrates the use of cellulase in detergents to reduce the harshness of cotton containing fabrics.
Another useful feature of cellulases in the treatment of textiles is their ability to recondition used fabrics by making their colors more vibrant. For example, repeated washing of cotton containing fabrics results in a greyish cast to the fabric which is believed to be due to disrupted and disordered fibrils, sometimes called “pills”, caused by mechanical action. This greyish cast is particularly noticeable on colored fabrics. As a consequence, the ability of cellulase to remove the disordered top layer of the fiber and thus improve the overall appearance of the fabric has been of value.
Because detergents, being a primary application of cellulase, operate generally under alkaline conditions there is a strong demand for cellulases which have excellent activity at pH 7-10. Well characterized fungal cellulases, such as those from
Humicola insolens
and
Trichoderma reesei
, perform adequately at neutral to low alkaline pH. Further, a number of enzymes that show cellulase activity at high alkaline pH have been isolated from Bacillus and other prokaryotes, see e.g., PCT Publication Nos. WO 96/34092 and WO 96/34108. Thus, both fungal and bacterial cellulases have been investigated thoroughly. However, a third group of cellulases, those isolated from Actinomycetes, have attracted only some attention. Wilson et al.,
Critical Reviews in Biotechnology
, Vol. 12, pp. 45-63 (1992), studied the cellulases produced by the
Thermomonospora fusca, Thermonomospora curvata
and
Microbispora bispora
and illustrated that many of these cellulases show broad pH profiles and good temperature stability. Similarly, Nakai et al.,
Agric. Biol. Chem.,
Vol. 51, pp. 3061-3065 (1987) and Nakai et al.,
Gene
, Vol. 65, pp. 229-238 (1988) exemplify the alkalitolerant cellulase casA from Streptomyces strain KSM-9 which also possesses an alkaline pH optimum and excellent temperature stability.
Despite knowledge in the art related to many cellulase compositions having desirable properties, including those from Actinomycetes, there is a continued need for new cellulases having a varying spectrum of characteristics which are useful in, for example, treating textiles, as a component of detergent compositions, in the treatment of pulp and paper, as an animal feed supplement, as a processing aid for baking and in the conversion of biomass. Applicants have discovered certain cellulases which have such a complement of characteristics and which are useful in such known applications of cellulase.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide for novel Actinomycete derived cellulase compositions having useful temperature and pH profiles for use in detergents.
It is a further object of the present invention to provide for novel Actinomycete derived cellulase compositions having useful characteristics for the treatment of textiles so as to produce desirable qualities in textile yams, fabrics and garments.
It is a further object of the present invention to provide for novel Actinomycete derived cellulases which have useful characteristics for use as an animal feed additive, as a baking aid, in the treatment of pulp and paper and in the reduction of biomass.
It is a further object of the present invention to provide for a method of producing cellulase compositions derived from such novel Actinomycetes.
It is yet a further object of the present invention to provide a DNA and amino acid sequence which facilitate industrial production of the novel cellulase compositions of the invention.
It is still a further object of the present invention to provide a novel cellulase having excellent properties for use in detergents, treating textiles, as a feed supplement and in pulp and paper manufacturing.
According to the present invention, a novel cellulase is provided which is obtainable from an Actinomycete or a derivative of said cellulase. Preferably, the cellulase of the invention comprises an amino acid sequence according to
FIG. 1
(SEQ ID NO: 1), or a derivative thereof having greater than 62% sequence identity, preferably greater than 75% sequence identity and more preferably greater than 90% sequence identity thereto.
According to another embodiment, a composition is provided comprising DNA encoding the cellulase of the invention. Preferably, the DNA encodes an amino acid sequence according to
FIG. 2
(SEQ ID NO:2), or a derivative thereof having greater than 72% sequence identity, preferably greater than 80% sequence identity and more preferably greater than 90% sequence identity thereto and cellulases produced thereby. The present invention further embodies DNA which hybridizes to a DNA probe taken from the DNA represented in
FIG. 2
under the appropriate conditions and cellulases produced thereby.
According to yet another embodiment of the invention, a method of transforming a suitable microorganism with DNA encoding a cellulase according to the invention is provided and a method of producing the cellulase according to the invention from that transformed microorganism.
In an especially preferred embodiment of the present invention, the mature cellulase is derived from Actinomycete and has a molecular weight of approximately 35 kD as measured on SDS-PAGE (referred to herein as the 35 kD cellulase). The mature approximately 35 kD cellulase has a calculated isoelectric point of about 4.5 and a pH optimum on CMC of about 6 at 40° C. and 6 or less at 60° C. The cellulase of the present invention showed higher activity at 60° C. than at 40° C. with broad activity ranges from at least pH 5 to pH 10.
REFERENCES:
patent: 5792641 (1998-08-01), Schulein et al.
patent: 5871550 (1999-02-01), Goedegebuur et al.
patent: 61-012282 (1986-01-01), None
patent: WO 96/00281 (1996-01-01), None
patent: WO 96/34108 (1996-10-01), None
patent: WO 96/34092 (1996-10-01), None
patent: WO 97/27363 (1997-07-01), None
Wilson, D.B. (1992) Crit. Rev. Biotechnol. 12(1/2), 45-63.
Lao, G. et al, “DNA sequences of three beta-1, 4 endoglucanase genes fromThermomonospora fusca”Journal of Bacteriology, vol. 173, No. 11, pp. 3397-3407 (1991).
Theberge M. et al, “Purification and characterization of an endoglucanase fromStreptomyces livdans66 and DNA sequence of the gene”,Applied and Environmental Biology, vol. 58, No. 3, pp. 815-820, (1992).
Nakai, R. et al., “Clo
Jones Brian E.
Van Der Kleij Wilhelmus A. H.
Van Solingen Piet
Weyler Walter
Faris Susan K.
Genencor International Inc.
Marcus-Wyner Lynn
Patterson Jr. Charles L.
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