Method of enhancing degradation of chitin

Abstract

The present invention provides a method of enhancing chitin degradation or weakening the structure of a chitin substrate comprising exposing chitin to a non-hydrolytic chitin binding protein (CBP). The invention further provides a method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase. Compositions, including fungicides, comprising non-hydrolytic CBPs and transgenic plants comprising exogenous nucleic acid molecules encoding a non-hydrolytic CBP are also provided.

Description

This invention will now be described in more detail in the following non-limiting Examples with reference to the drawings in which:

FIG. 1 shows scanning electron micrographs of β-chitin particles. The figure shows representative pictures of structures observed in the absence or presence of CBP21. Control particles (no CBP21 added) are shown in panels A and B (400× magnification) with close ups (5000× magnification) of the respective particles shown in panel E and F, respectively. Particles incubated with CBP21 are shown in panels C and D, with close ups (5000× magnification) shown in panels G and H, respectively. The black frame drawn on the 400× magnified images indicate the area targeted for the picture taken at 5000× magnification. The scale bars in panels A-D and E-H represent 50 and 5 μm, respectively.

FIG. 2 shows degradation of β-chitin in the absence or presence of CBP21. Reaction mixtures contained 0.1 mg/ml β-chitin, 0.2 μM enzyme and 5 μM CBP21, illustration purposes only. (A) ChiA (squares), ChiA+CBP21 (closed diamonds) or ChiA+CBP21 aded at t=48 h (open diamonds). (B) ChiB (squares), ChiB+CBP21 (closed diamonds), ChiB+CBP21 added at t=48 h (open diamonds) or ChiB+CBP21 added at t=216 h (squares connected by a dashed line; see text for details). C) ChiC (squares), ChiC+CBP21 (closed diamonds) or ChiC+CBP21 added at t=48 h (open diamonds) and D) 0.3 μM ChiG (squares) or 0.3 μM ChiG+5 μM CBP21 (diamonds).

FIG. 3 shows dose response effects for ChiC. Reaction mixtures contained 0.1 mg/ml β-chitin, 50 nM (A) or 5 nM (B) Chic and 500 (diamons), 50 (squares), 5 (triangles), 0.5 (crosses), 0.05 (hollow squares), 0.005 (hollow triangles) or 0 nM CBP21 (dotted line).

FIG. 4 shows synergistic effects in the degradation of β-chitin. The curves shows progress in degradation of β-chitin with various combinations of chitinases (as indicated by combinations of the letters A, B and C) and CBP21. The total enzyme concentration was always 50 nM, meaning that the reactions mixtures with one, two or three chitinases contained 50, 25 or 16.7 nM of each enzyme, respectively. The CBP21 concentration was 50 nM. For illustration purposes, the points are connected by dotted lines (single enzyme reactions), dashed lines (two-enzyme reactions) or solid lines (three-enzyme reactions). The effect of CBP21 may be evaluated by comparing curves with solid symbols (with CBP21) with curves with corresponding hollows symbols (same enzyme combination, no CBP21).

FIG. 5 shows the structure of CBP21. The side chains of all mutated residues are shown as sticks. Note that Ala152 and Gin161 were not expected to be involved in chitin binding.

FIG. 6 shows the degradation of β-chitin by ChiC in the presence of CBP21 mutants. Degradation of 0.1 mg/ml β-chitin with 50 nM ChiC in the presence of 50 nM CBP21 wild-type, Y54A, E55A, E60A, H114A, D182A, N185A, A152R or no CBP21 (indicated by a hyphen). Total product release is shown as black bars (24 h), grey bars (48 h) and light grey bars (120 h).

FIG. 7 shows multiple alignment of bacterial CBPs (Q8GBD4, Yersinia eneterocolitica; Q8ES33, Oceanobacillus iheyensis; Q8EHY2, Shewanella oneidensis; Q87FT0, Vibrio parahaemolyticus; Q7N4I5, Photorhabdus luminescens (subsp. laumondii); Q9CE94, Lactococcus lactis (subsp. lactis); Q9F9Q5*, Bacllus amyloliquefaciens; Q8Y4H4, Listeria monocytogenes; Q54501*, Streptomyces olivaceoviridis (CHB1); O83009*, Serratia marcescens (CBP21). CBPs marked with asterisks have been shown to bind chitin. The secondary structure elements of CBP21 are also shown and labelled. Arrows with residue numbers indicate residues mutated in Vaaje-Kolstad et al. (2005) JBC 280(12), 11313-11319. The multiple alignment was created with ClustalX and edited with T-COFFEE.

Claims

1. A method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic chitin binding protein (CBP).

2. A method of weakening the structure of a chitin substrate, comprising exposing chitin to a non-hydrolytic CBP.

3. The method of claim 1 or claim 2 wherein said chitin is α chitin, β chitin, g chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

4. The method of claim 1 or claim 2 which method comprises the steps of: (i) contacting said non-hydrolytic CBP and chitin under appropriate conditions so as to allow said non-hydrolytic CBP and chitin to interact or bind to each other(ii) incubating for sufficient time and under appropriate conditions to allow chitin weakening or enhancement of degradation to occur.

5. The method of claim 4 wherein the incubation step is carried out for 12 hours or more.

6. The method of claim 4 wherein said method is carried out at an appropriate temperance and pH.

7. The method of claim 4 wherein step (i) and/or (ii) are carried out at pH 6 and 6.5.

8. The method of claim 4 wherein said method is carried out without agitation (p16).

9. The method of claim 1 or claim 2 wherein non-hydrolytic CBP is, or corresponds to, or comprises a naturally occurring non-hydrolytic CBP derived from a microbial, eukaryotic or viral source, or is a functional variant thereof.

10. The method of claim 1 or claim 2 wherein the non-hydrolytic CBP consists of, or consists essentially of, or corresponds to, or comprises a family 33 carbohydrate binding module, or a functional variant thereof, or consists if, or consists essentially of, or corresponds to, or comprises a carbohydrate binding module from a family 33 carbohydrate binding molecule, or a functional variant thereof.

11. The method of claim 1 or claim 2 wherein the non-hydrolytic CBP is, or corresponds to, or comprises CBP21 of Serratia Marescens (SEQ ID NO: 1), ChbA of B. amyloliquefaciens, CHB1, 2 or 3 of Streptomyces, or CBP1 of Alteramonas, or a functional variant thereof.

12. The method of claim 11 wherein said functional variants display at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence similarly or identity with a naturally occurring non-hydrolytic CBP or CBM at the amino acid level.

13. The method of claim 9 wherein when said non-hydrolytic CBP is CBP21, then said functional variants retain one or more and preferably all of a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an asparagine residue at position 185.

14. The method of claim 9 wherein when the non-hydrolytic CBP is not CBP21, then said functional variants retain one or more of the residues at the positions corresponding to the CBP21 residues as defined in claim 13.

15. A method of enhancing chitin degradation comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase.

16. A method of degrading chitin comprising exposing chitin to a non-hydrolytic CBP and a chitin hydrolase.

17. The method of claim 15 or claim 16 wherein said chitin hydrolase is a chitinase enzyme.

18. The method of claim 17 wherein said chitinase enzyme is one or more of ChiA, ChiB, ChiC or ChiG.

19. The method of claim 15 or claim 16 wherein said chitin is α chitin, β chitin, γ chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

20. The method of claim 15 or claim 16 which method comprises the steps of: (i) contacting said non-hydrolytic CBP and chitin under appropriate conditions so as to allow said non-hydrolytic CBP and chitin to interact or bind to each other,(ii) incubating for sufficient time and under appropriate conditions to allow enhancement of degradation to occur.

21. The method of claim 20 wherein the incubation step is carried out for 12 hours or more.

22. The method of claim 20 wherein said method is carried out at an appropriate temperance and pH.

23. The method of claim 20 wherein step (i) and/or (ii) are carried out at pH 6 and 6.5.

24. The method of claim 20 wherein said method is carried out without agitation.

25. The method of claim 15 or claim 16 wherein non-hydrolytic CBP is, or corresponds to, or comprises a naturally occurring non-hydrolytic CBP derived from a microbial, eukaryotic or viral source, or is a functional variant thereof.

26. The method of claim 15 or claim 16 wherein the non-hydrolytic CBP consists of, or consists essentially of, or corresponds to, or comprises a family 33 carbohydrate binding module, or a functional variant thereof, or consists of, or consists essentially of, or corresponds to, or comprises a carbohydrate binding module from a family 33 carbohydrate binding molecule, or a functional variant thereof.

27. The method of claim 15 or claim 16 wherein the non-hydrolytic CBP is, or corresponds to, or comprises CBP21 of Serratia Marescens (SEQ ID NO: 1), ChbA of B. amyloliquefaciens, CHB1, 2 or 3 of Streptomyces, or CBP1 of Alteramonas, or a functional variant thereof.

28. The method of claim 27 wherein said functional variants display at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence similarly or identity with a naturally occurring non-hydrolytic CBP or CBM at the amino acid level.

29. The method of claim 25 wherein when said non-hydrolytic CBP is CBP21, then said functional variants retain one or more and preferably all of a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an aspartic acid residue at position 182 and an asparagine residue at position 185.

30. The method of claim 25 wherein the non-hydrolytic CBP is not CBP21, then said functional variants retain one or more of the residues at the positions corresponding to the CBP21 residues as defined in claim 29.

31. A method for identifying a non-hydrolytic CBP, or determining whether a protein is a non-hydrolytic CBP, wherein said putative non-hydrolytic CBP is exposed to chitin and the effect of this exposure on the putative non-hydrolytic CBP protein is assessed, and wherein if a weakening in chitin is observed or an enhancement in chitin degradation is observed, the protein is identified as being a non-hydrolytic CBP.

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. A composition comprising a non-hydrolytic CBP and a pharmaceutically or agriculturally acceptable carrier, diluent or excipient.

37. The composition of claim 36, wherein said compositions is a fungicide, nematocide, or an insecticide.

38. The composition of claim 36 further comprising other active ingredients such a a chitin hydrolase and/or a further fungicidal or pathicidal agent.

39. A method for treating or preventing disease in a plant, wherein said disease is caused by a chitin containing microorganism or pathogen, comprising contacting the chitin containing microorganism or pathogen or contacting the plant to be protected with an effective amount of a non-hydrolytic CBP p30.

40. The method for treating or preventing disease in an animal, wherein said disease is caused by a chitin containing microorganism or pathogen, comprising administering to a subject in need of such a treatment an effective amount of a non-hydrolytic CBP.

41. The method of claim 39 or 40, wherein said chitin containing microorganism or pathogen is a fungus, insect or nematode.

42. A method for combating or treating diseases caused by chitin containing plague organisms, which method comprises the administration to an organism of an agent which targets a non hydrolytic CBP.

43. The method of claim 42, wherein said organism is a plant or animal.

44. The method of claim 42, wherein the composition of claim 36 or claim 38 is administered.

45. A method for identifying residues in a non-hydrolytic CBP which are important for enabling an enhancement in chitin degradation or for weakening chitin, said method comprising the steps of: (i) mutating or otherwise altering one or more amino acid residues in the non-hydrolytic CBP, and(ii) testing the effect of the mutations or alterations on the property of enhancing chitin degradation or for weakening chitin.

46. The method of claim 45, wherein said method provides a method for generating a non-hydrolytic CBP with improved or increased ability to enhance the degradation of chitin, p34.

47. The method of claim 45 or claim 46, wherein said chitin is α chitin, β chitin, g chitin, amorphous chitin, colloidal chiting, chiti forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin.

48. A method of identifying, developing or producing a non-hydrolytic CBPs which can enhance chitin degradation or can weaken chitin, or variants thereof which have the ability to act on other substrates/polymers, said method comprising the steps of: (i) obtaining or otherwise providing a non-hydrolytic CBP,(ii) mutating or otherwise altering one or more amino acid residues in the non-hydrolytic CBP,(iii) assessing the ability of said mutated or altered non-hydrolytic CBP to degrade or to enhance the degradation of the substrate of interest or weaken the substrate of interest,(iv) identifying mutated or altered non-hydrolytic CBPs which display the desired properties.

49. The method of claim 48, wherein said substrate is a carbohydrate based structure or polymer.

50. The method of claim 49, wherein said substrate is α chitin, β chitin, g chitin, amorphous chitin, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, or a copolymer of chitin, cellulose or lignocellulose.

51. The method of claim 48, wherein said method is used to identify, develop or produce binding proteins which degrade or enhance the degradation of a different substrate to the non-hydrolytic CBP of step (i).

52. The method of claim 48 wherein the residues which are subjected to mutation or alteration are those which have been identified as being important for binding to chitin.

53. The method of claim 48 wherein the residues which are subject to mutation or alteration are those which have been identified as being important or essential for the enhancement of chitin degradation.

54. The method of claim 48 wherein the residues which are subjected to mutation or alteration are those present on the surface which binds to the substrate (the binding surface).

55. The method of claim 48 wherein the residues which are subjected to mutation or alteration are polar residues which are capable of forming specific polar interactions with the substrate.

56. The method of claim 48 wherein when said non-hydrolytic CBP is CBP21, then the residues which are subjected to mutation or alteration are one or more of: a tyrosine residue at position 54, a glutamic acid residue at position 55, a glutamic acid residue at position 60, a histidine residue at position 114, an asparagine residue at position 182 and an arginine residue at position 185.

57. The method of claim 48 wherein when the non-hydrolytic CBP is not CBP21, then the residues which are subjected to mutation or alteration are the residue at the positions corresponding to the CBP21 residues as defined in claim 56.

58. The method of claim 48, wherein the Y54 residue in CBP21 or its corresponding residue in non-hydrolytic CBPs that are not CBP21 is subject to mutation or alteration.

59. A method of identifying non hydrolytic CBP homologous or related proteins from other organisms or the same organism, said method comprising the steps of: (i) obtaining or otherwise providing one or more molecules of interest which are candidate homologous or related protein molecules, and(ii) testing or screening to assess whether said molecule of interest is a non-hydrolytic CBP which enhances chitin degradation or weakens chitin, or is a related protein with activities toward other substrates.

60. The method of claim 59, wherein said substrate is α chitin, β chitin, g chiting, amorphous chiting, colloidal chitin, chitin forms in which part of the N-acetylglucosamine sugars are deacetylated, chitosan, a copolymer of chitin, cellulose or lignocellulose.

61. The method of claim 59, wherein said molecules of interest used in step (i) are a library of molecules.

62. The method of claim 59, wherein said library of molecules is a library of non-hydrolytic CBP variants, which are to be tested for this activities on various substrates.

63. The method the claim 59, wherein step (ii) is carried out in accordance with the method of claim 1 or claim 2.

64. The method of claim 59, wherein step (ii) is carried out in accordance with the method of claim 15 or claim 16.

65. Substrate binding proteins or proteins which degrade or enhance the degradation of substrate identified, developed or produced by the method of claim 59.

66. (canceled)

68. A recombinant insect virus comprising a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP or a recombinant insect virus that expresses a naturally occurring non-hydrolytic CBP, under the control of an exogenous promoter.

69. The virus of claim 68 further comprising a nucleic acid sequence encoding a chitin hydrolytic enzyme such as a chitinase.

70. A method of increasing the infectivity of an insect virus comprising introducing a nucleic acid molecule that encodes an exogenous non-hydrolytic CBP into the genome of said insect virus and optionally introducing a nucleic acid molecule that encodes a chitin hydrolytic enzyme.

71. (canceled)

73. A method of treating a plant or animal comprising administering to said plant or animal an effective amount of the composition of claim 71.

74. A transgenic plant or parts thereof comprising an exogenous nucleic acid molecule comprising a sequence encoding a non-hydrolytic CBP.

75. The transgenic plant of claim 74, further comprising an exogenous nucleic acid molecule comprising a sequence encoding a chitin hydrolytic enzyme.

76. A method of modulating the level of non-hydrolytic CBP in a plant or a method of improving the resistance of a plant or a chitin containing pathogen comprising: a) introducing an expression cassette containing a polynucleotide encoding a non-hydrolytic CBP,b) culturing the plant cell under plant cell growing conditions, andc) inducing expression of the polynucleotide for a time sufficient to modulate the level of non-hydrolytic CBP in the plant.

77. The method of claim 76, wherein an expression cassette containing a polynucleotide encoding a chitin hydrolytic enzyme is also introduced.