Thermostable cellulase

Details Thermostable cellulase Thermostable cellulase

2001-10-23

2004-11-02

Rao, Manjunath (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S004000, C435S006120, C435S041000, C435S069100, C435S183000, C435S200000, C435S252300, C435S320100, C530S350000, C536S023200, C536S023400, C536S023700

Reexamination Certificate

active

06812018

ABSTRACT:

BACKGROUND OF THE INVENTION
Cellulases are enzymes that are capable of hydrolyzing cellulose. The products of the reaction include cellobiose and glucose which can in used for a variety of applications. For example, glucose obtained by cellulase catalyzed hydrolysis of plant cellulose can be fermented to produce ethanol which can be used as a fuel.
Cellulases can also be used in the de-inking and refining of recycled paper. Enzymes with high thermal stability are particularly useful in these applications because elevated temperatures are commonly used in these processes. Thermostable cellulases are also useful in the consumer products and food industries, for example, in extracting and clarifying juice from fruits or vegetables. Cellulases and particularly thermostable cellulases also have applications in the textile and laundry industries. For example, the enzyme can be used to remove microfibers from the surface of cotton garments (or other garments made of cellulose based fabric), thereby brightening the color and removing the dull look that comes with wear. Cellulases are also useful for cleaning garments, for example as additives to detergents and for producing a “stone-washed” effect on indigo dyed denim (see, U.S. Pat. No. 4,912,056).
The development of thermostable cellulases with improved stability and/or catalytic properties would provide advantages for the above-referenced applications and certain other applications. Therefore, a need exists for improved thermostable cellulases which can be easily produced.
SUMMARY OF THE INVENTION
The invention relates to polypeptides having thermostable cellulase activity. The polypeptides of the invention are variants of full-length or naturally occurring proteins that have thermostable cellulase activity and are readily produced in large quantities by expression in a host cell such as
Escherichia coli
. In one embodiment, the polypeptide is a variant of a glycosyl hydrolase of family 12 wherein one or more of the amino acid residues that are not part of the catalytic domain (e.g, one or more amino acid residues in the amino terminal hydrophobic domain and/or linker moiety) are deleted. In preferred embodiments, the polypeptide is derived from a thermophilic organism from a
Rhodotermus
species such as
R. marinus, R. obamae
, and
R. obamensis
; or a
Pyrococcus
species, including
P. abyssi, P. endeavori, P. furiosus, P. horikoshi, P. shinkai
, and
P. woesei
. In a more particular embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO:2 wherein one or more of the amino acid residues from position one to about position 40 are deleted. In even more particular embodiments, the polypeptide can have an amino acid sequence selected from residues 18-261 of SEQ ID NO:2, residues 19-261 of SEQ ID NO:2, residues 20-261 of SEQ ID NO:2, residues 21-261 of SEQ ID NO:2, residues 22-261 of SEQ ID NO:2, residues 23-261 of SEQ ID NO:2, residues 24-261 of SEQ ID NO:2, residues 25-261 of SEQ ID NO:2, residues 26-261 of SEQ ID NO:2, residues 27-261 of SEQ ID NO:2, residues 28-261 of SEQ ID NO:2, residues 29-261 of SEQ ID NO:2, residues 30-261 of SEQ ID NO:2, residues 31-261 of SEQ ID NO:2, residues 32-261 of SEQ ID NO:2, residues 33-261 of SEQ ID NO:2, residues 34-261 of SEQ ID NO:2, residues 35-261 of SEQ ID NO:2, residues 36-261 of SEQ ID NO:2, residues 37-261 of SEQ ID NO:2 or residues 38-261 of SEQ ID NO:2.
The polypeptides of the invention can have improved catalytic properties and/or stability relative to full-length enzyme. In one embodiment, the polypeptide has a half-life of at least about 3.5 hours at 90° C. In another embodiment, the polypeptide has a specific activity that is at least about two times greater than the specific activity of a protein consisting of the amino acid sequence of SEQ ID NO:2.
In particularly preferred embodiments, the polypeptide has the amino acid sequence of residues 18-261 of SEQ ID NO:2 or the amino acid sequence of residues 38-261 of SEQ ID NO:2.
The invention also relates to isolated nucleic acids which encode a polypeptide of the invention and to constructs which comprises an isolated nucleic acid of the invention that is operatively linked to one or more regulatory sequences.
The invention also relates to host cells which comprise an isolated nucleic acid or construct of the invention, and to a method of producing a polypeptide having thermostable cellulase activity. In one embodiment, the method comprises maintaining a host cell of the invention under conditions suitable for expression of the polypeptide that has thermostable cellulase activity.


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