Chimeric enzymes with improved cellulase activities
Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.
1. An isolated nucleic acid molecule encoding a chimeric CbhA polypeptide comprising domains from Clostridium thermocellum cellobiohydrolase A (CbhA) and Caldicellulosiruptor bescii cellobiohydrolase A (CelA) polypeptides, wherein the chimeric CbhA polypeptide has an amino acid sequence at least 95% identical to “SEQ ID NO:6,” and wherein the chimeric CbhA polypeptide has a cellulase activity at least 1.5-fold greater than the wild-type CbhA polypeptide.
2. The isolated nucleic acid molecule of claim 1 , wherein the chimeric CbhA polypeptide comprises the linker domain from the Caldicellulosiruptor bescii CelA polypeptide.
3. The isolated nucleic acid molecule of claim 1 , wherein the chimeric CbhA polypeptide has a cellulase activity at least 2-fold greater than the wild-type CbhA polypeptide.
4. The isolated nucleic acid molecule of claim 1 , wherein the chimeric CbhA polypeptide has an amino acid sequence at least 97% identical to SEQ ID NO:6.
5. The isolated nucleic acid molecule of claim 1 , wherein the chimeric CbhA polypeptide has the amino acid sequence of SEQ ID NO:6.
6. The isolated nucleic acid molecule of claim 1 , further comprising a promoter operably linked to the nucleic acid molecule.
7. The isolated nucleic acid molecule of claim 6 , wherein the promoter allows expression of the nucleic acid in a bacterial host cell.
8. An expression vector comprising the nucleic acid molecule of claim 1 .
9. An isolated host cell that expresses a recombinant polypeptide encoded by the nucleic acid molecule of claim 1 .
10. The host cell of claim 9 , wherein the cell is an E. coli cell.
11. An isolated chimeric CbhA polypeptide encoded by the nucleic acid molecule of claim 1 .
12. A method for degrading cellulose or lignocellulosic biomass, comprising contacting a cellulose containing material or lignocellulosic biomass with the isolated chimeric CbhA polypeptide of claim 11 .
13. A method for producing a biofuel from lignocellulosic biomass, comprising:
a) contacting the lignocellulosic biomass with an enzyme cocktail comprising the isolated chimeric CbhA polypeptide of claim 11 to generate sugars; and
b) converting the sugars to a biofuel by fermentation.
14. The method of claim 13 , wherein the enzyme cocktail further comprises an endoglucanase, a β-glucosidase, or both.