Thermal tolerant avicelase from acidothermus cellulolyticus

• US 20030108988A1
• Filed: 10/18/2002
• Published: 06/12/2003
• Est. Priority Date: 07/28/2001
• Status: Active Grant

• Current Assignee: Alliance For Sustainable Energy LLC
• Original Assignee: Alliance For Sustainable Energy LLC
• Inventors: Himmel, Michael E., Adney, William S., Vinzant, Todd B., Ding, Shi-You
• Granted Patent: US 7,538,200 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

An isolated polynucleotide molecule encoding a thermal stable AviIII polypeptide, said aviIII polypeptide comprising:

a) a sequence of SEQ ID NO: 1;

b) a sequence of SEQ ID NO: 3;

c) a sequence of SEQ ID NO: 4;

d) a sequence of SEQ ID NO: 5; and

e) a sequence having about 70% sequence identify with the sequence of a), b), c) or d).

Claims

16. An isolated polynucleotide molecule encoding a thermostable A viIII polypeptide, said A vi III polypeptide comprising:

a) a sequence of SEQ ID NO;

1;

b) a sequence of SEQ ID NO;

3;

c) a sequence of SEQ ID NO;

4;

d) a sequence of SEQ ID NO;

5;

e) a sequence having about 70% sequence identity with the sequence of a), b), C) or d).

17. The isolated polynucleotide molecule of claim 16 comprising a nucleic acid sequence having a 90% sequence identity to the sequence of SEQ ID NO:

2.

18. The isolated polynucleotide molecule of claim 16 comprising a nucleic acid sequence having about 80% sequence identity to the sequence of SEQ ID NO:

2.

19. The isolated polynucleotide molecule of claim 16, comprising a nucleic acid sequence having about 90% sequence identity to the nucleic acid sequence encoding the sequence of SEQ ID NO:

3.

20. The isolated polynucleotide molecule of claim 16, comprising a nucleic acid sequence having about 90% sequence identity to the nucleic acid sequence encoding the sequence of SEQ ID NO:

5.

21. The isolated polynucleotide molecule of claim 16, comprising a nucleic acid sequence having about 90% sequence identity to the nucleic acid sequence encoding the sequence of SEQ ID NO.1.

22. The isolated polynucleotide molecule of claim 16, further comprising a nucleic acid sequence encoding a heterologous protein in frame with the polynucleotide molecule of claim 1.

23. The isolated polynucleotide molecule of claim 22, wherein the heterologous protein is a peptide tag.

24. The isolated polynucleotide molecule of claim 22, wherein the peptide tag is 6-His, thioredoxin, hemaglutinin, GST, or OmpA signal tag.

25. The isolated polynucleotide molecule of claim 22, wherein the heterologous protein is a substrate targeting moiety.

26. The isolated polynucleotide molecule of claim 16, operably linked to a transcriptional or translational regulatory sequence.

27. The isolated polynucleotide molecule of claim 26, wherein the transcriptional or translational regulatory sequence comprises a transcriptional promoter or enchancer.

37. A vector comprising the polypeptide molecule of claim 27.

38. A host cell genetically engineered to express the polypeptide molecule of claim 27.

39. The host cell of claim 38, wherein the host cell is a plant cell.

40. The host cell of claim 38, wherein the host cell is a plant cell.

41. The host cell of claim 38, wherein the host cell is a bacterial cell.

42. The host cell of claim 38, wherein the host cell is a yeast.

47. A method for producing A viIII polupeptide, the method comprising;

incubating a host cell genectically engineered to express the polynucleotide molecule of claim 27.

48. The method of claim 47, further comprising the step of:

isolating the A viIII polypeptide from the incubated host cells.

49. The method of claim 47, wherein the host cell is a plant cell.

50. The method of claim 47, wherein the host cell is a bacterial cell.

51. The method of claim 47, wherein the host cell is genetically engineered to express a selectable marker.

52. The method of claim 47, wherein the host cell further comprises a polynucleotide molecule encoding one or more polypeptide molecules selected from the glycoside hydrolase family of proteins.

53. The method of claim 52, wherein the glycoside hydrolase is a thermostable glycoside hydrolase.

54. A set of amplification primers for amplification of a polynucleotide molecule encoding a thermostable A viIII, comprising:

two or more sequences comprising 9 or more contagious nucleic acids derived from the polynucleotide molecule of claim 28.

55. A probe for hybridizing to a polynucleotide encoding A viIII, comprising:

a sequence of 9 or more contiguous nucleic acids derived from a polynucleotide molecule of claim 28.

56. An assay method for the detection of a polynucleotide encoding a thermostable A viIII, comprising:

amplifying a nucleic acid sequence with a set of amplification primers comprising two or more sequence of 9 or more contiguous nucleic acids derived from the polynucleotide molecule of claims 28; and

correlating the amplified nucleic acid sequence with detected polynucleotide encoding a thermostable A viIII.

57. A method for assessing the carbohydrate degradation activity of A viIII comprising:

analyzing a carbohydrate degradation in the presence of A viIII and a carbohydrate degradation in the presence of A viIII and the agent of interest on a substrate; and

comparing the carbohydrate degradation in the presence of A viIII with the carbohydrate degradation in the abscence of A viIII.

58. A method for assessing the carbohydrate degradation activity of A viIII in the presence of an agent of interest comprising:

analying a carbohydrate degradation in the presence of A viIII and a carbohydrate degradation in the presence of A viIII and the agent of interest on a substrate exposed; and

comparing the carbohydration degradation in the A viIII treated substrate with the carbohydrate degradation in the A viIII treated substrate in the presence of the agent of interest.

59. The method of claim 58, wherein an increase in carbhydrate degradation activity in the presence of the agent of interest demonstarates stimulation of A viIII activity and wherein a drecrease in carbohydrate degradation activity demonstrates inhibition of A viIII activitiy.

60. The method of claim 58, wherein the carbohydrate is cellulose.

61. The method of claim 58, wherein the agent of interest is an antibody.

62. A method for reducing cellulose in a starting material, the method comprising:

administering to the starting material an effective amount of polypeptide molecule of claim 28.

63. The method of claim 62, further comprising administering a second polypeptide molecule selected from the glycoside hydrolase family of proteins.

64. The method of claim 62, wherein the starting material is agricultural biomass.

65. The method of claim 62, wherein the starting material is municipal solid waste.