Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-02-27
2003-05-20
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S006120, C536S023200
Reexamination Certificate
active
06566112
ABSTRACT:
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not Applicable.
BACKGROUND OF THE INVENTION
Cellulases are enzymes that hydrolyze 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.,
TIBTECH
5:255-261 (1987)). Cellulases 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. Cellulases are also known to be useful in detergent compositions for removing dirt, he., cleaning. For example, Great Britain Application Nos. 2,075,028, 2,095,275 and 2,094,826 illustrate improved cleaning performance with detergents that have incorporated cellulase. Additionally, Great Britain Application No. 1,368,599 illustrates the use of cellulase in detergents to reduce the harsh fell 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. This 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 found to be of value.
Because detergents, being a primary application of cellulase, operate generally under alkaline conditions there is a strong demand for cellulases that exhibit high 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. A number of enzymes demonstrating 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 meager attention. Wilson, et al.,
Critical Reviews in Biotechnology,
12:45-63 (1992), have studied cellulases produced by
Thermomonospora fusca, Thermonomospora curvata
and
Microbispora bispora
and have shown that many of these cellulases exhibit broad pH profiles and good temperature stability. Similarly, Nakai, et al.,
Agric. Biol. Chem.,
51: 3061-3065 (1987) and Nakai, et al.,
Gene,
65:229-238 (1988) have demonstrated the alkalitolerant cellulase casA from Streptomyces strain KSM-9. This cellulase possesses an alkaline pH optimum and excellent temperature stability.
Despite knowledge in the art related to many cellulase compositions having desirable properties, including some examples from Actinomycetes, there is a continued need for new cellulases having a varying spectrum of characteristics useful as, for example, textile treatments, components of detergent compositions, pulp and paper treatments, animal feed supplements, processing aids for baking, and biomass converters. Applicants have discovered cellulases which possess such a complement of characteristics and which are useful in such known applications of cellulases.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel cellulase compositions producible by Actinomycetes, derivatives of such cellulases, 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.
It is a further object of the present invention to provide for a method of producing cellulase compositions derived from such novel Actinomycetes via heterologous expression from recombinant host cells.
It is yet a further object of the present invention to provide a DNA and amino acid sequence which facilitate commercial 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 or a derivative is provided which is obtainable from an Actinomycete. Preferably, the cellulase of the invention comprises an amino acid sequence according to
FIG. 1
(SEQ ID NO:1), a fragment, or a derivative thereof, having greater than 50% sequence identity, preferably greater than 70% sequence identity and more preferably greater than 90% sequence identity to an active portion thereto.
According to another embodiment, a composition is provided comprising DNA encoding the cellulases of the invention. Preferably, the DNA encodes an amino acid sequence and comprises the nucleotide sequence as shown in
FIG. 2
(SEQ ID NO:2), a fragment, or a derivative thereof, having greater than 76% sequence identity, preferably greater than 80% sequence identity and more preferably greater than 90% sequence identity to a portion 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 a preferred embodiment of the present invention, a full-length cellulase is derived from Actinomycetes and has a molecular weight of approximately 36 kD as measured on SDS-PAGE (referred to herein as the 36 kD cellulase). The full-length, approximately 36 kD, cellulase has a calculated isoelectric point of about 5.9 and a pH optimum on CMC (carboxy methyl cellulose) of about 8 at 40° C. and 7 at 60° C. The cellulase of the present invention shows higher activity at 60° C. than at 40° C. with broad activity ranges from at least pH 5 to pH 10.
In an especially preferred embodiment, a cellulase of this invention is a truncated form of the full-length cellulase described above. Its molecular weight is approximately 25 kD.
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Perito, B., et al (1994) Acc. No. X73953.*
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Bibb et al., “Clo
Jones Brian E.
Van Der Kleij Wilhelmus A. H.
Van Solingen Piet
Weyler Walter
Genencor International Inc.
Genencor International Inc.
Patterson Jr. Charles L.
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