Enzymes having endoglucanase activity and methods of use thereof
OwnerJay M. Short, David E. Lam +1inventors
InventorsJay M. Short, David E. Lam, Eric J. Mathur
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Abstract
The invention relates to endoglucanases and to polynucleotides encoding the endoglucanases. In addition methods of designing new endoglucanases and method of use thereof are also provided. The endoglucanases have increased activity and stability at increased pH and temperature.
Claims92
An isolated nucleic acid comprising a sequence as set forth in SEQ ID NO:1 and variants thereof having at least about 50% identity to SEQ ID NO:1 and encoding a polypeptide having endoglucanase activity.
An isolated nucleic acid comprising at least 10 consecutive bases of SEQ ID NO:1, sequences substantially identical thereto, and sequences complementary thereto.
An isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID NO:2, and sequences substantially identical thereto.
An isolated nucleic acid encoding a polypeptide comprising at least 10 consecutive amino acids of a polypeptide having a sequence as set forth in of SEQ ID NO:2, and sequences substantially identical thereto.
A purified polypeptide having a sequence as set forth in SEQ ID NO:2 and sequences substantially identical thereto.
A purified antibody that specifically binds to a polypeptide comprising a sequence as set forth in SEQ ID NO:2, and sequences substantially identical thereto.
A purified antibody that specifically binds to a polypeptide having at least 10 consecutive amino acids of the polypeptides as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto.
A method of producing a polypeptide having a sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.
A method of producing a polypeptide comprising at least 10 amino acids of a sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto comprising introducing a nucleic acid encoding the polypeptide, operably linked to a promoter, into a host cell under conditions that allow expression of the polypeptide and recovering the polypeptide.
A method of generating a variant comprising:
- obtaining a nucleic acid comprising a sequence as set forth in SEQ ID NO:
- 1, sequences substantially identical thereto, sequences complementary thereto, fragments comprising at least 30 consecutive nucleotides thereof, and fragments comprising at least 30 consecutive nucleotides of the sequences complementary to SEQ ID NO:
- 1;
- and
- modifying one or more nucleotides in said sequence to another nucleotide, deleting one or more nucleotides in said sequence, or adding one or more nucleotides to said sequence.
A computer readable medium having stored thereon a nucleic acid sequence as set forth in SEQ ID NO:
- 1, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto.
A computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a nucleic acid sequence as set forth in SEQ ID NO:
- 1, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto.
A method for comparing a first sequence to a reference sequence wherein said first sequence is a nucleic acid sequence as set forth in SEQ ID NO:
- 1, and sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto comprising:
- reading the first sequence and the reference sequence through use of a computer program which compares sequences;
- and
- determining differences between the first sequence and the reference sequence with the computer program.
A method for identifying a feature in a sequence wherein the sequence is as set forth in SEQ ID NO:
- 1, sequences substantially identical thereto, or a polypeptide sequence as set forth in SEQ ID NO:
- 2, and sequences substantially identical thereto comprising:
- reading the sequence through the use of a computer program which identifies features in sequences;
- and
- identifying features in the sequences with the computer program.
A method of catalyzing the hydrolysis of cellulose comprising contacting a sample containing cellulose with a polypeptide selected from the group consisting of SEQ ID NO:
- 2 and sequences having at least 50% homology and having endoglucanase enzyme activity under conditions with facilitate the hydrolysis of the cellulose.
An assay for identifying functional polypeptide fragments or variants encoded by fragments of SEQ ID NO:
- 1, and sequences substantially identical thereto, which retain the enzymatic function of the polypeptides of SEQ ID NO:
- 2, and sequences substantially identical thereto, said assay comprising:
- contacting the polypeptide of SEQ ID NO:
- 2, and sequences substantially identical thereto, or polypeptide fragment or variant encoded by SEQ ID NO:
- 1, with a substrate molecule under conditions which allow said polypeptide or fragment or variant to function, and
- detecting either a decrease in the level of substrate or an increase in the level of the specific reaction product of the reaction between said polypeptide and substrate, wherein a decrease in the level of substrate or an increase in the level of the reaction product is indicative of a functional polypeptide or fragment or variant.
A nucleic acid probe comprising an oligonucleotide from about 10 to 50 nucleotides in length and having an area of at least 10 contiguous nucleotides that is at least 50% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:1 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 90% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:1 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 95% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:1 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A nucleic acid probe comprising an oligonucleotide from about 15 to 50 nucleotides in length and having an area of at least 15 contiguous nucleotides that is at least 97% complementary to a nucleic acid target region of the nucleic acid sequence set forth in SEQ ID NO:1 and which hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable target:probe duplex.
A polynucleotide probe for isolation or identification of endoglucanase genes having a sequence which is the same as or fully complementary to at least a portion of SEQ ID NO:1.
Dependencies are inferred from the claim text.