Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-08-19
2001-09-25
Weber, Jon P. (Department: 1643)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
Reexamination Certificate
active
06294366
ABSTRACT:
B. FIELD OF THE INVENTION
The present invention is directed to improved methods for treating cellulosic materials, including cotton-containing fabrics and non-cotton containing cellulose fabrics with novel truncated cellulase enzymes. In addition, this invention relates to novel truncated cellulase enzymes which exhibit cellulase activity, DNA constructs encoding the enzymes, cellulolytic agents comprising the enzymes, and detergent and water purifying or conditioning compositions containing the enzymes. In particular, this invention provides thermophilic cellulases isolated from a thermophilic anaerobic bacterial strain found in New Zealand. The cellulase genes from this organism are identified and sequenced, and the cellulases expressed from this bacterium are shown to be particularly useful in the abrasion of denim, and in the manufacture of clothing having a “stone wash” look. Most importantly, the cellulases of this invention possess unexpected proteolytic and chemical stability, as well as thermal and pH stability in hot alkaline solutions, thereby rendering them important to as laundry detergent additives in many industrial and home washing applications.
C. BACKGROUND OF THE INVENTION
During or shortly after their manufacture, cotton-containing fabrics can be treated with cellulase enzymes in order to impart desirable properties to the fabric. For example, in the textile industry, cellulase has been used to improve the feel and/or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, for imparting a stone washed appearance to cotton-containing denims and the like.
Clothing made from cellulose fabric, such as cotton denim, is stiff in texture due to the presence of sizing compositions used to ease manufacturing, handling and assembling of clothing items. It typically has a fresh dark dyed appearance. One desirable characteristic of indigo-dyed cloth is the alteration of dyed threads with white threads, which give denim a white on blue appearance.
After a period of extended wear and laundering, the clothing items, particularly denim, can develop in the clothing panels and on the seams, localized areas of variation in the form of a lightening, in the depth and density of color. In addition, a general fading of the clothes, some pucker in the seams and some wrinkling in the fabric panels can often appear. Additionally, after laundering, sizing is substantially removed from the fabric resulting in a softer feel. In recent years such a distressed or “stonewashed” look, particularly in denim clothing has become very desirable to a substantial proportion of the public.
Previous methods for producing the distressed look included stonewashing of a clothing item or items in a large tub with pumice stones having a particle size of about 1 by 1 inches and with smaller pumice particles generated by the abrasive nature of the process. Typically the clothing item is tumbled with the pumice while wet for a sufficient period such that the pumice abrades the fabric to produce in the fabric panels, localized abraded areas of lighter color and similar lightened areas in the seams. Additionally the pumice softens the fabric and produces a fuzzy surface similar to that produced by the extended wear and laundering of the fabric. This method also enhances the desired white on blue contrast described above.
The use of pumice stones has several disadvantages, including overload damage to the machine motors, mechanical damage to transport mechanisms and washing drums, environmental waste problems from the grit produced and high labor costs associated with the manual removal of the stones from the pockets of the garments.
In view of the problems associated with pumice stones in stonewashing, cellulase solutions are used as a replacement for the pumice stones under agitating and cascading conditions, i. e., in a rotary drum washing machine, to impart a “stonewashed” appearance to the denim.
Cellulases are enzymes which hydrolyze cellulose (&bgr;-1,4-D-glucan linkages) and produce as primary products glucose, cellobiose, cello-oligosaccharides and the like. Cellulases are produced by a number of microorganisms and comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (CBH), endoglucanases (EG), and &bgr;-glucosidases (BG). Enzymes within these classifications can be separated into individual components. The complete cellulase system comprising CBH, EG, and BG components synergistically act to convert crystalline cellulose to glucose.
A problem with the use of complete cellulase compositions from previously described microorganism sources for stonewashing dyed denim is the incomplete removal of colorant caused by redeposition or “backstaining” of some of the dye back onto the cloth during the stonewashing process. In the case of denim fabric, this causes recoloration of the blue threads and blue coloration of the white threads, resulting in less contrast between the blue and white threads and abrasion points (i.e., a blue on blue look rather than the preferred white on blue.)This redeposition is objectionable to some users.
Some cellulases are used commercially even though they result in backstaining because of their higher activity in denim material. Either high specific activity or a high level of purity results in a higher degree of abrasion in a significantly shorter processing time and therefore is preferable to commercial denim processors.
Attempts to reduce the amount of redeposition of dye included the addition of extra chemicals or enzymes, such as surfactants, proteases, or other agents, into the cellulase wash to help disperse the loosened dye. In addition, processors have used less active whole cellulase, along with extra washings. However this results in additional chemical costs and longer processing times. Finally the use of enzymes and stones together leave the processor with all the problems caused by the use of the stones alone. Accordingly, it would be desirable to find a method to prevent redeposition of colorant during stonewashing with cellulases.
There have been previous attempts to prevent backstaining. Patent WO 92/06221 of Genecor pertains to backstaining and indicates that the cellulose biohydralase (CBH) found in fungal cellulases is largely responsible for strength loss of the fabric and that a 5 to 1 ratio of endoglucanase to CBH is desirable. WO 96/23928, also to Genencor, relates to use of a truncated cellulase core enzyme. Both of these references emphasize the use of buffers to stabilize the cellulase solution in the wash environment. In the art it is recognized that cellulase activity is pH dependent. Most cellulases will exhibit cellulolytic activity within an acidic to neutral pH range, and the pH of an unbuffered cellulase solution could be outside the range required for cellulolytic activity. This can be undesirable and requires the addition of reagents to lower the pH of the denim following the wash cycle increasing the processing expense.
Applications of cellulases for textile processing and in commercial detergents demand proteins which are stable under highly alkaline conditions in the presence of surfactants as well as elevated temperatures.
D. BRIEF DESCRIPTION OF THE INVENTION
Microorganisms from New Zealand hot springs are a recognized potential source of alkalophilic and thermophilic enzymes. We have examined numerous of these microorganisms isolated from thermal pools for their cellulase activity under alkaline conditions. The approach used was to grow the isolated bacterial cultures on cotton in order to enrich for strains that contain cellulase activity. Selected strains were grown on a larger scale and culture supernatants were then individually screened for the desired stone-wash effect. A particular strain of unknown species, but most closely resembles those in the Caldicellulosiruptor genus and which has been called by us, Tok7B.1, was identified from this testing. Further investigation resulted in the discovery, in accord with this invention, of six differ
Anderson Paige
Bergquist Peter
Daniels Roy
Farrington Graham K.
Gibbs Moreland David
Clariant Finance (BVI) Limited
Hanf Scott E
Pfeiffer Hesna J.
Weber Jon P.
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