Enzymatic hydrolysis of fucoidan
阅读说明：本技术 褐藻糖胶的酶促水解 (Enzymatic hydrolysis of fucoidan ) 是由 A.博拉斯顿 C.J.维克斯 O.E.萨拉马-阿尔伯 K.T.阿贝 于 2019-07-24 设计创作，主要内容包括：来自嗜冷单胞菌属的菌种的岩藻糖酶,包括P5AFcnA岩藻糖酶和P19DFcnA岩藻糖酶。还有与之相关的方法、系统、组合物等,包括在被这样的岩藻糖酶水解后具有降低的平均分子量的褐藻糖胶组合物。(Fucosidases from species of the genus psychrophila, including P5AFcnA fucosidases and P19DFcnA fucosidases. Also related are methods, systems, compositions, etc., including fucoidan compositions having a reduced average molecular weight after hydrolysis by such fucoidans.)
1. A composition comprising P5AFcnA fucosidase in aqueous solution and added fucoidan.
2. A composition comprising a P19DFcnA fucosidase in aqueous solution and added fucoidan.
3. The composition of claim 1 or 2, wherein the added fucoidan is a raw fucoidan composition.
4. The composition of any one of claims 1 to 3, wherein the composition has a pH of about 6.5-9.0.
5. The composition of any one of claims 1 to 4, wherein the temperature of the composition is about 15-40 ℃.
6. The composition according to any one of claims 1 to 5, wherein the composition is under the conditions: the conditions are such that the fucosidase is capable of hydrolyzing glycosidic bonds in fucoidan.
7. A fucosidase produced by a eukaryotic entity, the fucosidase being encoded by a gene sequence according to any one of: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, wherein less than 5% of the codons are substituted.
8. A fucosidase produced by a species of the genus Psychromonas (psychromyonas species) encoded by a gene sequence according to any one of: SEQ ID NO.1 or SEQ ID NO.3 wherein at least 1 codon and less than 5% of the codons are substituted.
9. A fucosidase produced by a species of psychrophila that is encoded by a gene sequence according to any one of: SEQ ID NO.2 or SEQ ID NO.4, wherein less than 5% of the codons have been substituted.
10. A fucosidase produced by a prokaryotic entity other than a psychrophilum species, the fucosidase being encoded by a gene sequence according to any one of: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No.4, wherein less than 5% of the codons are substituted.
11. A fucosidase produced by a eukaryotic entity, the fucosidase comprising an amino acid sequence according to any one of: SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8, wherein less than 5% of the amino acids are substituted.
12. A fucosidase produced by a species of psychrophila comprising an amino acid sequence according to any one of: SEQ ID NO.5 or SEQ ID NO.7 wherein at least 1 amino acid and less than 5% of the amino acids are substituted.
13. A fucosidase produced by a species of psychrophila comprising an amino acid sequence according to any one of: SEQ ID NO.6 or SEQ ID NO.8 wherein less than 5% of the amino acids are substituted.
14. A fucosidase produced by a prokaryotic entity other than a species of the genus psychrophila, the fucosidase comprising an amino acid sequence according to any one of: SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No.8, wherein less than 5% of the amino acids are substituted.
15. The fucosidase of claim 10 or 14 wherein the prokaryotic entity is escherichia coli (e.coli).
16. A method, comprising: fucosidases of the genus psychrophila are used to selectively enzymatically hydrolyze fucoidan in an artificial aqueous solution.
17. The method of claim 16, wherein the psychrophila fucosidase is encoded by a gene sequence according to any one of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, wherein less than 5% of the codons are substituted.
18. The method of claim 16, wherein the psychrophila fucosidase is encoded by a gene sequence according to any one of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, wherein less than 3% of the codons are substituted.
19. The method of claim 16, wherein the psychrophila fucosidase is encoded by a gene sequence according to any one of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, wherein less than 1% of the codons are substituted.
20. The method of claim 16, wherein the psychromonas fucosidase comprises an amino acid sequence according to any one of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, wherein less than 5% of the amino acids are substituted.
21. The method of claim 16, wherein the psychromonas fucosidase comprises an amino acid sequence according to any one of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, wherein less than 3% of the amino acids are substituted.
22. The method of claim 16, wherein the psychromonas fucosidase comprises an amino acid sequence according to any one of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, wherein less than 1% of the amino acids are substituted.
23. The method of claim 16, wherein the fucoidan is a fucoidan obtained from at least one of: ascophyllum nodosum (Adenocystis utriculalis), Ascophyllum nodosum (Ascophyllum nodosum), Chorda funiculosum (Chorda filum), Cystosemilabies marina, Antarctic bull algae (Durvilia antarctica), Ecklonia kurome (Ecklonia kurome), Ecklonia megalophylla (Eisenia bicyclis), Fucus evanescens (Fucus evanescens), Fucus vesiculosis (Fucus vesiculosis), Hizikia fusiformis (Hizikia fusiforme), Ostrinia Elongata (Himanthia Elongata), Laminaria clarkia (Sacchararia crassifolia), Laminaria japonica (Laminaria japonica), Laminaria japonica (Laminariaceae), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestris (Laminaria), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestita) and Laminaria japonica (Eucalypta), Laminaria japonica (Eucalypta) and Laminaria japonica (Eucalypta) strains, Eucalypta) and Laminaria japonica, Eucalypta japonica, Eucalypti (Eucalypti, Laminaria japonica, Eucalypta) plants (Eucalypti, Laminaria) and Laminaria japonica, Eucalypti (Eucalypti, Laminaria) and Laminaria (Eucalypti, Laminaria) are, Laminaria) and Laminariaceae.
24. The method of claim 16, wherein the fucoidan is a fucoidan obtained from at least one of: saccharomyces japonica, Laminaria hyperborea, Macrocystis grandis (Macrocystis pyrifera) and Chorda filium (Chorda filum).
25. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by at least 5%.
26. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 10%.
27. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 20%.
28. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 30%.
29. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 40%.
30. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 50%.
31. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 60%.
32. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 70%.
33. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 80%.
34. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 90%.
35. The process of any one of claims 16-24, wherein the average molecular weight of said fucoidan is reduced by about 95%.
36. The method of any one of claims 16-24, wherein at least 5% of glycosidic linkages in said fucoidan are hydrolyzed.
37. The method of any one of claims 16-24, wherein at least 10% of the glycosidic linkages in the fucoidan are hydrolyzed.
38. The method of any one of claims 16-24, wherein at least 20% of glycosidic linkages in said fucoidan are hydrolyzed.
39. The method of any one of claims 16-24, wherein at least 30% of glycosidic linkages in said fucoidan are hydrolyzed.
40. The method of any one of claims 16-24, wherein at least 40% of glycosidic linkages in the fucoidan are hydrolyzed.
41. The method of any one of claims 16-24, wherein at least 50% of the glycosidic linkages in the fucoidan are hydrolyzed.
42. The method of any one of claims 16-24, wherein at least 60% of the glycosidic linkages in the fucoidan are hydrolyzed.
43. The method of any one of claims 16-24, wherein at least 70% of the glycosidic linkages in the fucoidan are hydrolyzed.
44. The method of any one of claims 16-24, wherein at least 80% of glycosidic linkages in said fucoidan are hydrolyzed.
45. The method of any one of claims 16-24, wherein at least 90% of glycosidic linkages in the fucoidan are hydrolyzed.
46. The method of any one of claims 16-24, wherein at least 95% of glycosidic linkages in the fucoidan are hydrolyzed.
47. A process for obtaining a desired lower molecular weight fucoidan composition relative to a starting fucoidan composition by enzymatically hydrolyzing fucoidan in the starting fucoidan composition, the process comprising:
providing the starting fucoidan composition and a psychrophila fucosidase in an aqueous solution; incubating the starting fucoidan composition and the psychrophila fucosidase under conditions sufficient to hydrolyze fucoidan in the starting fucoidan composition to produce a hydrolyzed fucoidan and a hydrolysis residue molecule; and
separating the hydrolyzed fucoidan from the hydrolyzed residual molecules and the fucosidase to obtain a fucoidan composition of a desired molecular weight.
48. The method of claim 47, wherein the fucosidase is encoded by a gene sequence according to one of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
49. The method of claim 47, wherein the fucosidase comprises an amino acid sequence according to one of: SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
50. The method of claim 47, wherein said fucoidan is obtained from at least one of: ascophyllum nodosum (Adenocystis utriculalis), Ascophyllum nodosum (Ascophyllum nodosum), Chorda funiculosum (Chorda filum), Cystosemilabies marina, Antarctic bull algae (Durvilia antarctica), Ecklonia kurome (Ecklonia kurome), Ecklonia megalophylla (Eisenia bicyclis), Fucus evanescens (Fucus evanescens), Fucus vesiculosis (Fucus vesiculosis), Hizikia fusiformis (Hizikia fusiforme), Ostrinia Elongata (Himanthia Elongata), Laminaria clarkia (Sacchararia crassifolia), Laminaria japonica (Laminaria japonica), Laminaria japonica (Laminariaceae), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestris (Laminaria), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestita) and Laminaria japonica (Eucalypta), Laminaria japonica (Eucalypta) and Laminaria japonica (Eucalypta) strains, Eucalypta) and Laminaria japonica, Eucalypta japonica, Eucalypti (Eucalypti, Laminaria japonica, Eucalypta) plants (Eucalypti, Laminaria) and Laminaria japonica, Eucalypti (Eucalypti, Laminaria) and Laminaria (Eucalypti, Laminaria) are, Laminaria) and Laminariaceae.
51. The method of claim 47, wherein said fucoidan is obtained from at least one of: saccharomyces japonica, Laminaria hyperborea, Macrocystis grandis (Macrocystis pyrifera) and Chorda filium (Chorda filum).
52. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by at least 5%.
53. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 10%.
54. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 20%.
55. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 30%.
56. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 40%.
57. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 50%.
58. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 60%.
59. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 70%.
60. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 80%.
61. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 90%.
62. The process of any one of claims 47-51, wherein the average molecular weight of said fucoidan is reduced by about 95%.
63. The method of any one of claims 47-51, wherein at least 5% of the glycosidic linkages in said fucoidan are hydrolyzed.
64. The method of any one of claims 47-51, wherein at least 10% of the glycosidic linkages in said fucoidan are hydrolyzed.
65. The method of any one of claims 47-51, wherein at least 20% of the glycosidic linkages in said fucoidan are hydrolyzed.
66. The method of any one of claims 47-51, wherein at least 30% of the glycosidic linkages in said fucoidan are hydrolyzed.
67. The method of any one of claims 47-51, wherein at least 40% of the glycosidic linkages in said fucoidan are hydrolyzed.
68. The method of any one of claims 47-51, wherein at least 50% of the glycosidic linkages in said fucoidan are hydrolyzed.
69. The method of any one of claims 47-51, wherein at least 60% of the glycosidic linkages in the fucoidan are hydrolyzed.
70. The method of any one of claims 47-51, wherein at least 70% of the glycosidic linkages in said fucoidan are hydrolyzed.
71. The method of any one of claims 47-51, wherein at least 80% of the glycosidic linkages in said fucoidan are hydrolyzed.
72. The method of any one of claims 47-51, wherein at least 90% of the glycosidic linkages in said fucoidan are hydrolyzed.
73. The method of any one of claims 47-51, wherein at least 95% of the glycosidic linkages in the fucoidan are hydrolyzed.
74. An expression vector comprising an expressible P5AFcnA fucosidase gene.
75. An expression vector comprising an expressible P19DFcnA fucosidase gene.
76. An expression vector comprising an expressible fucosidase gene of the genus psychrophila.
77. An expression vector comprising an expressible gene sequence according to any one of: SEQ ID NO.1 or SEQ ID NO.3, wherein less than 5% of the codons are substituted.
78. An expression vector comprising an expressible gene sequence according to any one of: SEQ ID NO.2 or SEQ ID NO.4, wherein less than 5% of the codons have been substituted.
79. The expression vector of claims 74-78, wherein said expression vector is a plasmid.
80. The expression vector of claims 74-78, wherein said expression vector is a prokaryotic expression plasmid.
81. The expression vector according to claim 80, wherein the prokaryotic expression plasmid is configured for expression in E.
82. The expression vector of claims 74-78, wherein said expression vector is a eukaryotic expression plasmid.
83. A method of making an enzyme, the method comprising: expressing a gene according to any one of claims 74 to 82 and collecting the enzyme expressed by the gene.
84. A method of making an enzyme, the method comprising: expressing a gene sequence according to claims 71 to 82 and collecting the enzymes expressed from such gene.
85. The method of claim 83 or 84, wherein the enzyme is a fucosidase.
86. A method of using an expressed fucosidase, the method comprising: providing a fucosidase obtained from the expression vector of any one of claims 74-82, and combining the expressed fucosidase with fucoidan under conditions selected for the fucosidase to hydrolyze the fucoidan.
87. The method of claim 86, wherein said fucoidan is provided as a raw fucoidan composition.
88. The method of claim 86 or 87, wherein said expressed fucosidase hydrolyzes glycosidic linkages within the fucoidan.
Background
Fucoidan (including fucoidan) is a sulfated polysaccharide. In general, this means that fucoidan is a molecule composed of many sugar groups, and also has a sulfur atom attached to the sugar group. The main glycosyl group is called "fucose", which is a sugar having 6 carbon atoms and of formula C6H12O5The sugar of (1). "fucoidan" (or fucoidan) indicates fucoidan derived from brown algae (seaweed). Fucoidan may be present alone or in mixtures of other sugars, for example in mixtures of sugars such as xylose, galactose, glucose, glucuronic acid and/or mannose. These other sugars may be extracted from the seaweed or other sources along with the fucoidan. Although fucoidan is currently derived from natural sources such as brown algae (seaweed), sea cucumbers, etc. as mentioned herein, "fucoidan" includes polymer molecules having the chemical and structural motifs of fucoidan as discussed herein, regardless of the ultimate source or sources of the fucoidan.
Fucoidan can be obtained from a variety of brown algae species, including but not limited to: ascophyllum nodosum (Adenocystis utriculalis), Ascophyllum nodosum (Ascophyllum nodosum), Chorda funiculosum (Chorda filum), Cystosemilabies marina, Antarctic bull algae (Durvilia antarctica), Ecklonia kurome (Ecklonia kurome), Ecklonia megalophylla (Eisenia bicyclis), Fucus evanescens (Fucus evanescens), Fucus vesiculosis (Fucus vesiculosis), Hizikia fusiformis (Hizikia fusiforme), Ostrinia Elongata (Himanthia Elongata), Laminaria clarkia (Sacchararia crassifolia), Laminaria japonica (Laminaria japonica), Laminaria japonica (Laminariaceae), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestris (Laminaria), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestita) and Laminaria japonica (Eucalypta), Laminaria japonica (Eucalypta) and Laminaria japonica (Eucalypta) strains, Eucalypta) and Laminaria japonica, Eucalypta japonica, Eucalypti (Eucalypti, Laminaria japonica, Eucalypta) plants (Eucalypti, Laminaria) and Laminaria japonica, Eucalypti (Eucalypti, Laminaria) and Laminaria (Eucalypti, Laminaria) are, Laminaria) and Laminariaceae. These exemplary species are all from the class Phaeophyceae (Phaeophyceae) and most of these species belong to the families: fucales (Fucales) and Laminariaceae (Laminariaceae).
Fucoidan, including fucoidan, has been shown to be effective as a barrier for preventing, inhibiting, and treating the formation of fibrous adhesions. It also finds use in the treatment of other related diseases and disorders.
Thus, there has been an unmet need for an improved process for preparing fucoidan compositions having a desired molecular weight distribution. Previous work discusses the use of fucosidase (called substrate-specific MfFcnA enzyme) from flavobacterium strain SW5 (Colin et al, 2006) for the hydrolysis of certain fucoidans. The methods, systems, etc. of the present invention are directed to, among other advantages, providing enzymes capable of hydrolyzing selected fucoidan compositions, and methods of using these enzymes to obtain desired molecular weight fucoidan distribution compositions from starting fucoidan compositions.
Disclosure of Invention
Methods, systems, compositions, etc. are provided for obtaining and utilizing fucoidans that are capable of enzymatically hydrolyzing fucoidans, including fucoidans in a starting fucoidan composition, to obtain a desired lower molecular weight distribution of the fucoidan relative to the starting fucoidan composition. Embodiments herein include fucosidase genes and fucosidase amino acid sequences: it can be used for producing or synthesizing fucosidase, and producing fucoidan composition with reduced average molecular weight fucoidan after hydrolyzing by fucosidase. The process of hydrolyzing the starting fucoidan composition using the fucosidase produced provides for time-based control of fucoidan substrate selectivity and during enzymatic hydrolysis.
The method, system and the like of the invention comprise: desired fucoidan compositions comprising a desired molecular weight distribution are obtained from a starting fucoidan composition, as well as compositions comprising such desired fucoidan molecular weight distributions, and methods of using such compositions. In one aspect, the method comprises:
the compositions, systems, methods, and the like of the present invention provide compositions that can include P5AFcnA fucosidase and/or P19DFcnA fucosidase in an aqueous solution with added fucoidan. The fucoidan added may be a raw fucoidan composition that may have a pH of about 6.5-9.0, a temperature of about 15-40 ℃, and be under conditions such that the fucosidase can hydrolyze glycosidic bonds in the fucoidan.
In certain embodiments, the compositions, systems, methods, and the like of the present invention include enzymes, such as fucoidans produced from prokaryotic or eukaryotic entities encoded by a gene sequence according to any one of SEQ ID NOs 1 to 4, or encoded by a gene sequence based on such SEQ ID NOs: wherein at least 0, 1, 2, 3, 4 or 5 codons and less than 1%, 2%, 3%, 4% or 5% of the codons are substituted. In further embodiments, the compositions, systems, and methods, etc., of the present invention include a fucosidase produced from a prokaryotic or eukaryotic entity, the fucosidase comprising an amino acid sequence according to any one of SEQ ID nos. 5 to 9 wherein at least 0, 1, 2, 3, 4, or 5 amino acids and less than 1%, 2%, 3%, 4%, or 5% of the amino acids are substituted. The prokaryotic entity may be a species of the genus Psychromonas (Psychromonas species) or Escherichia Coli (Escherichia Coli).
The method can comprise the following steps: fucoidan is enzymatically hydrolyzed using the psychrophila fucoidans or other fucoidans discussed herein in an aqueous solution other than in the psychrophila species (e.g., in an artificial solution contained in an artificial container). Fucoidan may be fucoidan, and may be obtained from at least one of: ascophyllum nodosum (Adenocystis utriculalis), Ascophyllum nodosum (Ascophyllum nodosum), Chorda funiculosum (Chorda filum), Cystosemilabies marina, Antarctic bull algae (Durvilia antarctica), Ecklonia kurome (Ecklonia kurome), Ecklonia megalophylla (Eisenia bicyclis), Fucus evanescens (Fucus evanescens), Fucus vesiculosis (Fucus vesiculosis), Hizikia fusiformis (Hizikia fusiforme), Ostrinia Elongata (Himanthia Elongata), Laminaria clarkia (Sacchararia crassifolia), Laminaria japonica (Laminaria japonica), Laminaria japonica (Laminariaceae), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestris (Laminaria), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestita) and Laminaria japonica (Eucalypta), Laminaria japonica (Eucalypta) and Laminaria japonica (Eucalypta) strains, Eucalypta) and Laminaria japonica, Eucalypta japonica, Eucalypti (Eucalypti, Laminaria japonica, Eucalypta) plants (Eucalypti, Laminaria) and Laminaria japonica, Eucalypti (Eucalypti, Laminaria) and Laminaria (Eucalypti, Laminaria) are, Laminaria) and Laminariaceae. In certain embodiments, the fucoidan or fucoidan is obtained from at least one of saccharomyces japonica, Laminaria japonica (Laminaria hyperborea), macropystis pyrifera, and chlorella filifera (Chorda filum). Hydrolysis may result in a decrease in the average molecular weight of the fucoidan of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Hydrolysis may cause at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the glycosidic linkages in the hydrolyzed fucoidan to be hydrolyzed.
Also provided are methods for obtaining a desired low molecular weight fucoidan composition relative to a starting fucoidan composition by enzymatically hydrolyzing the fucoidan in the starting fucoidan composition. The method can comprise the following steps:
providing a starting fucoidan composition and a psychrophila fucosidase in an aqueous solution;
incubating the starting fucoidan composition and the psychrophilum fucosidase under conditions sufficient to hydrolyze fucoidan in the starting fucoidan composition to produce a hydrolyzed fucoidan and a hydrolysis residue molecule; and
the hydrolyzed fucoidan is separated from the hydrolysis residue molecules and the fucosidase to obtain a desired molecular weight fucoidan composition.
As discussed herein, the fucosidase may be encoded by a gene sequence and/or may comprise an amino acid sequence, e.g. according to one of SEQ ID nos. 1 to 8, and as discussed herein, an alginate/alginate raw material composition may be obtained. The average molecular weight and/or glycosidic linkages present in the alginate/algin composition may be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.
Also provided are expression vectors comprising an expressible P5AFcnA fucosidase gene, a P19DFcnA fucosidase gene, or other sequences discussed herein, as well as enzymes and fucoidans discussed herein and expressed from and typically collected and purified from such expression vectors.
These and other aspects, features and embodiments are set forth in this application, including the following detailed description and drawings. All embodiments, aspects, features, etc. may be mixed and matched, combined, and arranged in any desired manner, unless explicitly stated otherwise.
Drawings
Figure 1 depicts a flow diagram of an exemplary process for obtaining a lower molecular weight fucoidan composition relative to a starting fucoidan composition by enzymatic hydrolysis.
Figure 2A provides an example of a C-PAGE gel showing that the fucoidan composition extracted from saccharomyces Japonica is hydrolyzed by P5AFcnA fucosidase and P19DFcnA fucosidase, and that the fucoidan composition extracted from saccharomyces Japonica is not hydrolyzed by MfFcnA fucosidase.
Fig. 2B provides an example of a C-PAGE gel showing that a fucoidan composition extracted from Laminaria japonica aresch (Laminaria hyperborea) is hydrolyzed by P5AFcnA fucosidase and P19DFcnA fucosidase, and that such a fucoidan composition is not hydrolyzed by MfFcnA fucosidase.
Figure 2C provides an example of a C-PAGE gel showing that a fucoidan composition extracted from Macrocystis pyrifera is hydrolyzed by P5AFcnA fucosidase and P19DFcnA fucosidase, and that this fucoidan composition is not hydrolyzed by MfFcnA fucosidase.
The figures, including the flow charts, present exemplary embodiments of the disclosure. Actual implementations of the systems, methods, etc. herein may include other features or steps not shown in the figures. The examples set forth herein illustrate embodiments of the systems, methods, etc., in one or more forms, and such examples should not be construed as limiting the scope of the disclosure in any way. The embodiments herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, for example, in the following detailed description.
Detailed Description
The methods, systems, compositions, etc., herein relate to fucosidases: the fucosidase is capable of enzymatically hydrolyzing the raw fucoidan composition to obtain a desired lower molecular weight distribution of fucoidan relative to the raw fucoidan composition. Thus, the fucoidans discussed herein are useful for selectively and controllably cleaving fucoidan to reduce its size, thereby selectively and controllably decreasing the molecular weight distribution of the fucoidan in the starting composition to provide a modified fucoidan composition comprising a desired lower molecular weight distribution of the modified fucoidan.
Certain embodiments of the methods and the like are illustrated as examples in the figures with fucoidan as the fucoidan gum. The coding regions for the corresponding natural and synthetic fucosidases are discussed as are the fucosidases gene sequences and the composition of the fucosidases amino acid sequences. The lower molecular weight fucoidan composition obtained as an enzymatic hydrolysate of the starting fucoidan composition has a lower average molecular weight distribution relative to the starting fucoidan distribution. This shift in molecular weight distribution may be accompanied by changes in the dispersibility and shape of the distribution.
Turning to the development of certain fucosidases discussed herein, fucoidan-soluble psychrophila species SW5A and SW19D were isolated from brown macroalgae collected from the coast of the gulf of cabbago (cabbioy Bay) and the Beach of vilows (willowbeach) (victoria, wengowski, columbia, canada). Genomic DNA was extracted from cultured psychrophila species SW5A and SW19D and the next generation sequencing of their genomes was performed on the Illumina MiSeq platform. The genome was annotated to identify putative genes encoding carbohydrate-active enzymes. In the course of this annotation, the fucosidase gene sequences encoding the P5AFcnA and P19DFcnA enzymes were identified in the genomes of the species psychrophilus sp.sw5a and psychrophilus sp.sw19d.
Fucosidase gene sequences encoding the P5AFcnA and P19DFcnA enzymes were obtained from the genomes of the species psychrophilus sp.sw5a and psychrophilus sp.sw19d. FromSynthetic fucosidase genes encoding the P5AFcnA and P19DFcnA enzymes were ordered, codon optimized for expression in e.coli, and pre-inserted into e.coli expression plasmid pET28 a. These constructs were designated pET28a _ P5AFcNA and pET28a _ P19 DFcNA. The sequence fidelity of the constructs was confirmed by bidirectional DNA sequencing. Fucosidase is expressed in an escherichia coli overexpression system with a hexahistidine tag. To purify the recombinant protein, cells were harvested, chemically lysed and centrifuged to separate cell debris from the cell lysate supernatant. The protein was then purified from the lysate by immobilized nickel affinity chromatography. The purified protein was dialyzed against 0.5M NaCl buffer into 20mM Tris pH 8.5 and concentrated to 29. mu.M for later use. The coding region sequences of the fucosidase gene for natural and synthetic P5AFcNA fucoidans are shown in tables 1A and 1B, respectively, as SEQ ID NO.1 and 2, respectively. The coding region sequences of the fucosidase gene for natural and synthetic P19DFcNA fucoidans are shown in tables 1C and 1D, respectively, as SEQ ID NO.3 and 4, respectively. The amino acid sequences of natural and synthetic P5AFcnA fucoidases are shown in tables 1E and 1F, respectively, as SEQ ID nos. 5 and 6, respectively. The amino acid sequences of natural and synthetic P19DFcNA fucoidases are shown in tables 1G and 1H, respectively, as SEQ ID No.7 and 8, respectively. In some embodiments, the nucleic acid sequences set forth herein are controllably expressed in a suitable expression vectorColumn, https:// en. wikipedia. org/wiki/Expression _ vector. Such expression vectors may be optimized for prokaryotic or eukaryotic expression, and may include plasmids, expression viruses, cell-free systems, and the like, as desired. The expression vector may further include selected primers, an origin of replication, and the like, as necessary.
The fucosidase gene sequence SEQ ID NO.1 shown in Table 1A is hereinafter referred to as the native P5AFcNA gene sequence. Hereinafter, the fucosidase gene sequence SEQ ID NO.2 shown in Table 1B is referred to as a synthetic P5AFcNA gene sequence. Hereinafter, the fucosidase gene sequence SEQ ID NO.3 shown in Table 1C is referred to as a natural P19DFcNA gene sequence. Hereinafter, the fucosidase gene sequence SEQ ID NO.4 shown in Table 1D is referred to as a synthetic P19DFcNA gene sequence. The synthetic sequences were optimized for expression of the enzyme with E.coli. The amino acid sequence SEQ ID NO.5 shown in Table 1E is hereinafter referred to as the natural P5AFcNA amino acid sequence. The amino acid sequence SEQ ID NO.6 shown in Table 1F is hereinafter referred to as the synthetic P5AFcNA amino acid sequence. The amino acid sequence SEQ ID NO.7 shown in Table 1G is hereinafter referred to as the natural P19DFcNA amino acid sequence. The amino acid sequence SEQ ID NO.8 shown in Table 1H is hereinafter referred to as the synthetic P19DFcNA amino acid sequence.
Enzymatic hydrolysis of fucoidan
The flow diagram of figure 1 depicts an exemplary process [100] for obtaining a desired lower molecular weight fucoidan composition relative to the starting fucoidan composition by enzymatic hydrolysis of the starting fucoidan composition, i.e., for reducing the average molecular weight of the fucoidan in the composition. In this exemplary non-limiting method, the method includes: providing a starting fucoidan composition in the form of an [110] aqueous solution (i.e., an artificial, usually buffered solution); incubating a starting fucoidan composition in the form of a solution with a fucosidase to produce a solution comprising a hydrolyzed fucoidan composition, a fucosidase, and a hydrolyzed residual molecule [120 ]; and separating [130] the solution comprising the hydrolyzed fucoidan composition from the hydrolyzed residual molecules and the fucosidase to obtain the desired lower molecular weight fucoidan composition.
Providing [110] a starting fucoidan composition can include: providing an fucoidan/raw fucoidan composition extracted from at least one of: ascophyllum nodosum (Adenocystis utriculalis), Ascophyllum nodosum (Ascophyllum nodosum), Chorda funiculosum (Chorda filum), Cystosemilabies marina, Antarctic bull algae (Durvilia antarctica), Ecklonia kurome (Ecklonia kurome), Ecklonia megalophylla (Eisenia bicyclis), Fucus evanescens (Fucus evanescens), Fucus vesiculosis (Fucus vesiculosis), Hizikia fusiformis (Hizikia fusiforme), Ostrinia Elongata (Himanthia Elongata), Laminaria clarkia (Sacchararia crassifolia), Laminaria japonica (Laminaria japonica), Laminaria japonica (Laminariaceae), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestris (Laminaria), Laminaria japonica (Eucalyptus japonica), Laminaria japonica (Eucalyptus sylvestita) and Laminaria japonica (Eucalypta), Laminaria japonica (Eucalypta) and Laminaria japonica (Eucalypta) strains, Eucalypta) and Laminaria japonica, Eucalypta japonica, Eucalypti (Eucalypti, Laminaria japonica, Eucalypta) plants (Eucalypti, Laminaria) and Laminaria japonica, Eucalypti (Eucalypti, Laminaria) and Laminaria (Eucalypti, Laminaria) are, Laminaria) and Laminariaceae. In certain embodiments, the fucoidan may be obtained from at least one of Saccharomyces japonica, Laminaria japonica (Laminaria hyperborea), Macrocystis pyrifera, and Chorda filum.
Providing a starting fucoidan composition in the form of an [110] aqueous solution may comprise: the starting fucoidan composition is provided in the form of a solution at a fucoidan concentration of between about 0.01% w/v to about 30% w/v. Providing a starting fucoidan composition in the form of an [110] aqueous solution may comprise: the starting fucoidan composition is provided in the form of a solution at a fucoidan concentration of between about 0.05% w/v to about 10% w/v. Providing a starting fucoidan composition in the form of an [110] aqueous solution may comprise: the starting fucoidan composition is provided in the form of a solution at a fucoidan concentration of between about 0.1% w/v to about 5% w/v.
Providing [110]]The starting fucoidan composition in the form of an aqueous solution may include: providing a starting fucoidan composition in the form of a solution comprising at least one of: sodium dihydrogen phosphate and phosphoric acidDisodium, trisodium phosphate, phosphate buffer, phosphate buffered saline, trimethylglycine buffer, borate buffer, Tris buffer (also known as Tris buffer), sodium chloride, TrizmaTMBuffer, β -hydroxy-4- (2-hydroxyethyl) piperazine-1-propanesulfonic acid (also known as HEPPSO) buffer, piperazine-l, 4-bis (2-hydroxypropanesulfonic acid) dihydrate (also known as POPSO) buffer, Triethanolamine (TEA) buffer, 3- [4- (2-hydroxyethyl) piperazin-1-yl]Propane-1-sulfonic acid (EPPS) buffer, glycine buffer, N- (2-hydroxyethyl) piperazine-N' - (4-butanesulfonic acid) (HEPBS) buffer, 2- [4- (2-hydroxyethyl) piperazin-1-yl]Ethanesulfonic acid (HEPES) buffer, [ tris (hydroxymethyl) methylamino]Propanesulfonic acid (TAPS) buffer, 2-amino-2-methyl-1, 3-propanediol (also known as AMPD) buffer, N-tris (hydroxymethyl) methyl-4-amino-butanesulfonic acid (also known as TABS) buffer, N- (l, 1-dimethyl-2-hydroxyethyl) -3-amino-2-hydroxy-propanesulfonic Acid (AMPSO) buffer, 2- (cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (cyclohexylamino) -2-hydroxy-1-propane-sulfonic acid (CAPSO) buffer, 2-amino-2-methyl-1-propanol (also known as AMP) buffer, 3- (cyclohexylamino) -1-propane-sulfonic acid (CAPS) buffer, and mixtures thereof, 4- (cyclohexylamino) -1-butanesulfonic acid (CABS) buffer, citrate phosphate buffer, sodium carbonate-bicarbonate buffer, and ammonium bicarbonate. The buffer solution may have a pH of, for example, weakly acidic to weakly basic, e.g., a pH of about 5.5-10.5, 6.5-9.0, 7.5-8.8, or 8.0-8.5.
Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the starting fucoidan composition is incubated with a fucosidase of the genus psychrophila. Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the raw fucoidan composition is incubated with recombinant fucosidase produced by E.coli. Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the starting fucoidan composition is incubated with at least one or both of P5AFcnA and P19DFcnA fucoidases (e.g., synthetically produced non-natural P5AFcnA and P19DFcnA fucoidases). Incubating the raw fucoidan composition with a fucosidase [120] can include incubating the raw fucoidan composition with such a fucosidase: the fucosidase is translated from its fucosidase gene or modified fucosidase gene by a species of escherichia coli or psychrophila or any other suitable prokaryotic or eukaryotic host/product species. The methods herein can include producing an enzyme by expressing a fucosidase gene sequence of at least one of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, wherein less than 5% of the codons are substituted. Incubating the raw fucoidan composition with a fucosidase [120] can include incubating the raw fucoidan composition with a fucosidase encoded by a sequence that: in this sequence, at least one, three or five but less than 1%, 3% or 5% of the codons or amino acids of the natural P5AFcnA sequences SEQ ID No.1 and 5, respectively, are substituted. Incubating the raw fucoidan composition with a fucosidase [120] can include incubating the raw fucoidan composition with a fucosidase encoded by a sequence that: in this sequence, less than 1%, 3% or 5% of the codons or amino acids of the synthetic P5AFcnA sequences SEQ ID No.2 and 6, respectively, are substituted. Incubating the raw fucoidan composition with a fucosidase [120] can include incubating the raw fucoidan composition with a fucosidase encoded by a sequence that: in this sequence at least one, three or five but less than 1%, 3% or 5% of the codons or amino acids of the natural P19DFcnA sequences SEQ ID No.3 and 7, respectively, are substituted. Incubating the raw fucoidan composition with a fucosidase [120] can include incubating the raw fucoidan composition with a fucosidase encoded by a sequence that: in this sequence, less than 1%, 3% or 5% of the codons of the synthetic P19DFcnA sequences SEQ ID No.4 and 8, respectively, were substituted.
Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the raw fucoidan composition-fucosidase mixture is maintained at a temperature of between about 0 ℃ and about 60 ℃, between about 15 ℃ and 40 ℃, between about 20 ℃ and 30 ℃, e.g., about 23 ℃, about 25 ℃, and about 27 ℃.
Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the mixture is agitated in at least one form, such as by stirring, shaking, or mixing the raw fucoidan composition-fucosidase mixture, for example for at least 1 minute to the entire incubation period.
Incubating the starting fucoidan composition with a fucosidase [120] can comprise: the raw fucoidan composition-fucosidase mixture is incubated for between about 30 minutes to about 300 hours, between about 1 hour to about 100 hours, between about 2 hours to about 50 hours, between about 3 hours to about 24 hours, such as about 5 hours, about 10 hours, about 15 hours, and about 20 hours.
Incubating the starting fucoidan composition with a fucosidase [120] can comprise: incubating the starting fucoidan composition-fucosidase mixture until a desired molecular weight is obtained in the fucoidan composition. Incubating the starting fucoidan composition with a fucosidase [120] can comprise: incubating the starting fucoidan composition-fucosidase mixture until the average molecular weight obtained in the fucoidan composition is reduced by at least about 5% or 10%, and the average molecular weight obtained in the fucoidan composition is reduced by at least about 20%, for example, until the average molecular weight obtained in the fucoidan composition is reduced by about 40%, about 50%, or about 60%. Incubating the starting fucoidan composition with a fucosidase [120] can comprise: incubating the raw fucoidan composition-fucosidase mixture until at least 5% or 10% of the glycosidic linkages in the fucoidan in the raw fucoidan composition are hydrolyzed and at least 20% of the glycosidic linkages in the fucoidan in the raw fucoidan composition are hydrolyzed, e.g., until about 40%, about 50%, and about 60% of the glycosidic linkages are hydrolyzed.
Separating [130] the solution comprising the hydrolyzed fucoidan composition from the hydrolyzed residual molecules and the fucosidase can comprise: prior to separation, the fucosidase in the solution comprising the hydrolyzed fucoidan composition is quenched with a quenching agent to terminate the hydrolysis. Quenching the fucosidase in the solution may include: the solution was rendered alkaline. Making the solution alkaline may include: the pH of the solution is increased to about 9-14. Making the solution alkaline may include: at least one of NaOH, KOH, and LiOH is added to the solution. The fucosidase in the quench solution may also or alternatively comprise: the solution is heated to between 60-100 c, for example to about 80 c or above. Quenching the fucosidase in the solution may also or alternatively comprise: precipitating the fucosidase with a precipitating agent (such as at least one of ethanol, isopropanol, propanol, and methanol).
Separating [130] the hydrolyzed fucoidan composition from the hydrolyzed residual molecules and the fucosidase may comprise: the solution was diafiltered through a Tangential Flow Filtration (TFF) filter. Diafiltration may include: diafiltering the solution comprising the hydrolyzed fucoidan composition with at least one of distilled water, a salt solution, and a buffer solution. Diafiltration may include: the solution containing the hydrolyzed fucoidan composition is diafiltered through a TFF filter having a molecular weight cut-off less than the desired molecular weight in the desired fucoidan composition. Diafiltration may include: the starting fucoidan composition in the solution is diafiltered through a TFF filter having a molecular weight cut-off of any one of 5kDa, 10kDa, 30kDa, 50kDa, 70kDa, or 100 kDa. Separating [130] the hydrolyzed fucoidan composition from the hydrolyzed residual molecules and the fucosidase may comprise at least one of centrifugation, filtration, and sedimentation.
Examples
The above process discussed in FIG. 1 was applied to separate raw fucoidan compositions extracted from Saccharomyces japonica, Ascophyllum nodosum (Ascophyllum nodosum), Pelvetia Canaliculata (Pelvetia Canalicula), Fucus vesiculosus (Fucus vesiculosus), Laminaria arctica (Laminaria hyperborea) and Macrocystis pyrifera (Macrocystis pyrifera). The fucosidases used were the MfFcnA fucosidases previously discussed (Colin et al, 2006) as well as the P5AFcnA and P19DFcnA fucosidases obtained as described above. The raw fucoidan composition was dissolved at about 0.1 to 0.2% w/v in 20mM Tris buffer pH 8.5 with 0.5M NaCl. Separately, 29 μ M solutions of MfFcnA, P5AFcnA, or P19DFcnA in the same Tris buffer, pH 8.5, with 0.5M NaCl were added to isolate the solution of the raw fucoidan composition to a final concentration of 10 μ M fucosidase. The raw fucoidan composition-fucosidase mixture was all incubated at 25 ℃ on an orbital shaker at 100rpm for 15 hours.
After 15 hours, the occurrence of hydrolysis in each individual raw fucoidan composition-fucosidase mixture was analyzed using a carbohydrate-polyacrylamide gel electrophoresis (C-PAGE) technique: mu.L of the hydrolyzed fucoidan composition-fucosidase mixture was mixed with 10. mu.L of the loading dye (10% v/v glycerol, 0.01% w/v aqueous solution of bromophenol blue). The resulting mixture was loaded onto a 24% w/v C-PAGE gel run on ice, first at 100 volts for 15 minutes, second at 150 volts for 20 minutes, and finally at 200 volts for 20 minutes.
The results of the C-PAGE analysis are shown in Table 2 below, in which the occurrence of hydrolysis was evaluated. Examples of the absence and presence of hydrolysis on C-PAGE gels are shown in FIG. 2A, FIG. 2B and FIG. 2C. In the table, "+" indicates that hydrolysis of the fucoidan composition was observed on the gel, "-" indicates that no hydrolysis of the fucoidan composition was observed on the gel, and "+/-" indicates that minimal hydrolysis was observed.
Source of raw fucoidan composition
MfFcnA
P5AFcnA
P19DFcnA
Saccharina japonica
-
+
+
Ascophyllum nodosum (Ascophyllum nodosum)
+
-
-
Ditch antler (Pelvetia Canalicula)
+
-
-
Fucus vesiculosus (Fucus vesiculosus)
+/-
-
-
Laminaria japonica (Laminaria hyperborea)
-
+
+
Macrocystis (Macrocystis pyrifera)
-
+
+
TABLE 2 enzymatic hydrolysis of fucoidan using three different fucoidans
The method discussed above and in fig. 1 was applied under different conditions to separate raw fucoidan compositions extracted from different sources: kelp (Macrocystis pyrifera), Chorda filicina (Chorda filum), Ascophyllum nodosum (Ascophyllum nodosum), Laminaria japonica (Laminaria hyperborea), Saccharina japonica and Fucus vesiculosus (Fucus vesiculosus). The raw fucoidan composition was dissolved at 0.25% w/v and incubated with 1 μ M of the same fucosidase at room temperature for 40 hours. The same Tris buffer composition was used, the pH was adjusted to 8.0.
The analysis was performed on C-PAGE and the results are shown in Table 3 below, in which the occurrence of hydrolysis was evaluated. In the table, "+" indicates that hydrolysis of the fucoidan composition was observed on the gel, "-" indicates that no hydrolysis of the fucoidan composition was observed on the gel, and "+/-" indicates that minimal hydrolysis was observed.
TABLE 3 enzymatic hydrolysis of fucoidan using three different fucoidans
Table 2 and table 3 in conjunction with fig. 2A, fig. 2B, and fig. 2C show that P5AFcnA fucosidase and P19DFcnA fucosidase exhibit different enzyme activities compared to the MfFcnA fucosidase discussed previously. P5AFcNA fucoidases and P19DFcNA fucoidases are capable of enzymatically degrading fucoidan extracted from Laminaria japonica (Laminaria hyperborea) and kelp (Macrocystis pyrifera) and degrading Saccharina japonica to a greater extent, which MfFcNA fucoidases cannot do. The difference in results between two separate experiments accounts for the fact that: fucoidan extracted from different sources, even from the same species, may show subtle differences in the framework structure, which may depend on geographical or seasonal variations.
The present application further relates to fucoidan compositions prepared according to the various elements of the methods, systems, etc. discussed herein, as well as methods of using the compositions and systems and apparatuses configured to perform the methods herein and obtain the desired fucoidan compositions, etc.
List of reference numerals:
100 is used in a process for obtaining a desired low molecular weight fucoidan composition relative to a starting fucoidan composition by enzymatically hydrolyzing the starting fucoidan composition.
110 provides the starting fucoidan composition in the form of an aqueous solution.
120 incubating the starting fucoidan composition in solution with a fucosidase to produce a solution comprising a hydrolyzed fucoidan composition, a fucosidase, and a hydrolyzed residual molecule.
130 separating the solution containing the hydrolyzed fucoidan composition from the hydrolyzed residual molecules and the fucosidase to obtain the desired molecular weight of the fucoidan composition.
All terms used herein are used in their ordinary sense unless context or definition clearly indicates otherwise. In addition, as used in the specification, "or" includes "and" vice versa, unless explicitly stated otherwise. Non-limiting terms are not to be construed as limiting (e.g., "including," "having," and "containing" generally mean "including, but not limited to") unless expressly specified otherwise or clear from the context. The singular forms (including in the claims such as "a", "an" and "the") include plural references unless expressly stated otherwise or the context clearly dictates otherwise.
Unless otherwise indicated, an adjective such as "substantially" and "about" modifying a condition or relationship characteristic of one or more features of an embodiment herein is indicative that the condition or feature is defined as being within an acceptable tolerance for operation of the embodiment for the intended application.
The scope of the methods, compositions, systems, etc., of the present invention includes both means plus function and step plus function concepts. However, unless the word "means" is specifically recited in the claims, the claims should not be construed as a relationship representing "means plus function", whereas in the case where the word "means" is specifically recited in the claims, the claims are to be construed as a relationship representing "means plus function". Similarly, unless the word "step" is specifically recited in a claim, the claims should not be construed as meaning a "step plus function" relationship, whereas where the word "step" is specifically recited in a claim, the claims are to be construed as meaning a "step plus function" relationship.
From the foregoing, it will be appreciated that, although specific embodiments have been discussed herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the discussion herein. Accordingly, the systems and methods, etc., include such modifications and all permutations and combinations of the subject matter presented herein, and are limited only by the appended claims or other claims that have sufficient support in the discussion and drawings herein.
Sequence listing
<110> ARC medical instruments GmbH
<120> enzymatic hydrolysis of fucoidan
<130> 1946-023-05
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<213> modified bacterium
<400> 33
agcgactttg ttgcgatgct gcgtagcgtg gatccggagc tgaccattgc ggttaactac 60
<210> 34
<211> 60
<212> DNA
<213> modified bacterium
<400> 34
gatcaacact atttcaccga cgataacggt gaatacctgt atgtggacag cgatggcctg 60
<210> 35
<211> 60
<212> DNA
<213> modified bacterium
<400> 35
gacgatgagg acgaaagcga ttacaagatc gttaaacacg tggttaccaa cgagtatatg 60
<210> 36
<211> 60
<212> DNA
<213> modified bacterium
<400> 36
gactttacca acggtcatgt gaccccgctg ggtcgtggcg cgccgccgaa cagctgggcg 60
<210> 37
<211> 60
<212> DNA
<213> modified bacterium
<400> 37
tacgaggaat ataccattcc ggacatgatc gaggttccgt gggaaaccta cgatggtagc 60
<210> 38
<211> 60
<212> DNA
<213> modified bacterium
<400> 38
aagtatgcgc tgaaacacgg ctggttcccg attcgtaaca gctggagcgg cagcaaggcg 60
<210> 39
<211> 60
<212> DNA
<213> modified bacterium
<400> 39
gaactgatgt tcgacgtgga gcaggcgtac cgttttgtgc gtaccgttac cgatggtggc 60
<210> 40
<211> 60
<212> DNA
<213> modified bacterium
<400> 40
gcggcgatga cctggagcac cacccaagac aacggttata tgaccgcgga tgaaatgagc 60
<210> 41
<211> 60
<212> DNA
<213> modified bacterium
<400> 41
atcatgattg agatcagcaa ccgtatgacc cagaccccga agccggatta cagcgtgtat 60
<210> 42
<211> 42
<212> DNA
<213> modified bacterium
<400> 42
gaacgtccga aaggcgcgta cctggttagc gagatcgaat aa 42
<210> 43
<211> 60
<212> DNA
<213> bacterium
<400> 43
atgttaatcg gctgtggtgg aagtagtgca aatcaaactc aatctacgag cgaccaggac 60
<210> 44
<211> 60
<212> DNA
<213> bacterium
<400> 44
tcatctgaag ttaatgaaac ggtgagctta gacagcgaat atacagctaa ttggatggct 60
<210> 45
<211> 60
<212> DNA
<213> bacterium
<400> 45
ggtgcttggg gcataactca acgcgtagac ggcggttata aacttgatgc ttctgtagaa 60
<210> 46
<211> 60
<212> DNA
<213> bacterium
<400> 46
acaggaaaat atgattgggt cgctggtgca gaagaaattg ttgaaaatat tccttcagct 60
<210> 47
<211> 60
<212> DNA
<213> bacterium
<400> 47
ggttatgtaa ttacatcatt tactcatccc gcccatggtt ttttatatac tttaagagat 60
<210> 48
<211> 60
<212> DNA
<213> bacterium
<400> 48
aatgaaaacg tagatgttgc tgctattcat cctgatatgg tgccttcttt agaaaatgaa 60
<210> 49
<211> 60
<212> DNA
<213> bacterium
<400> 49
aaaatvatvt ttgatgttat taatgtatat aaatctgccg gtaagaaagt attattatat 60
<210> 50
<211> 60
<212> DNA
<213> bacterium
<400> 50
ttgaatacgg caggtcctac tcatgctgca gatagaaact cccccgaaat tcaagatgca 60
<210> 51
<211> 60
<212> DNA
<213> bacterium
<400> 51
tgggatgatt atgtaaatac gaattggaat ggggatcatg gtgcagcttg gaggaatcta 60
<210> 52
<211> 60
<212> DNA
<213> bacterium
<400> 52
gttgaaggtt acgctaaaag attcaaaggt ttagttgatg ggttttggtt agataattca 60
<210> 53
<211> 60
<212> DNA
<213> bacterium
<400> 53
aaaaacatgg ctggaggtca aaaagaaatc cctgaatttc ttgcaatgct tagagatatt 60
<210> 54
<211> 60
<212> DNA
<213> bacterium
<400> 54
gatccgtcat ttgcaatagg cgtaaattat gaaactcatt attttgaaga tgaagatggt 60
<210> 55
<211> 60
<212> DNA
<213> bacterium
<400> 55
aactacttaa aagtagcatc agatagcata gatgataacg atgatcgtga atacaaaata 60
<210> 56
<211> 60
<212> DNA
<213> bacterium
<400> 56
ataaaacatg tagtgaccaa tgaatatatg gactttacta atggccatgt cactccaatg 60
<210> 57
<211> 60
<212> DNA
<213> bacterium
<400> 57
ggacaaggtg ccccacctaa ttcttggggg tatgaagaat atacaatccc acacatgatt 60
<210> 58
<211> 60
<212> DNA
<213> bacterium
<400> 58
gaaaaacctt gggatagtgt tgatggtaat cattatgcac ttatgcatgg ttggttccct 60
<210> 59
<211> 60
<212> DNA
<213> bacterium
<400> 59
atcagattct cttggagcgg ttcaggtgct gagctcatgt ttgaaactga acaagcttat 60
<210> 60
<211> 60
<212> DNA
<213> bacterium
<400> 60
cggtttgtcc gcacgatcac tgatggtggt gcagcaatga catggtcaac cactcaaaaa 60
<210> 61
<211> 60
<212> DNA
<213> bacterium
<400> 61
aagggttata tgtcacgaga tgaaatggat ataatgattg aaattaacaa cagaatgaca 60
<210> 62
<211> 60
<212> DNA
<213> bacterium
<400> 62
caagccccta aactagatta cgaagcttat gaaagaccag aaggggcata tttggttggt 60
<210> 63
<211> 12
<212> DNA
<213> bacterium
<400> 63
gaaatagaat aa 12
<210> 64
<211> 60
<212> DNA
<213> modified bacterium
<400> 64
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
<210> 65
<211> 60
<212> DNA
<213> modified bacterium
<400> 65
atgaaccaga cccaaagcac cagcgaccag gatagcagcg aggtgaacga aaccgttagc 60
<210> 66
<211> 60
<212> DNA
<213> modified bacterium
<400> 66
ctggacagcg aatataccgc gaactggatg gcgggtgcgt ggggcattac ccaacgtgtg 60
<210> 67
<211> 60
<212> DNA
<213> modified bacterium
<400> 67
gacggtggct acaagctgga tgcgagcgtt gaaaccggta aatatgattg ggtggcgggc 60
<210> 68
<211> 60
<212> DNA
<213> modified bacterium
<400> 68
gcggaggaaa ttgttgagaa catcccgagc gcgggttacg tgatcaccag cttcacccac 60
<210> 69
<211> 60
<212> DNA
<213> modified bacterium
<400> 69
ccggcgcacg gctttctgta taccctgcgt gacaacgaga acgtggatgt tgcggcgatt 60
<210> 70
<211> 60
<212> DNA
<213> modified bacterium
<400> 70
cacccggaca tggtgccgag cctggagaac gaaaagatca tcttcgatgt gatcaacgtt 60
<210> 71
<211> 60
<212> DNA
<213> modified bacterium
<400> 71
tacaagagcg cgggtaagaa agttctgctg tatctgaaca ccgcgggtcc gacccatgcg 60
<210> 72
<211> 60
<212> DNA
<213> modified bacterium
<400> 72
gcggaccgta acagcccgga aattcaggat gcgtgggacg attacgtgaa caccaactgg 60
<210> 73
<211> 60
<212> DNA
<213> modified bacterium
<400> 73
aacggtgacc acggtgcggc gtggcgtaac ctggtggagg gttatgcgaa gcgtttcaaa 60
<210> 74
<211> 60
<212> DNA
<213> modified bacterium
<400> 74
ggtctggttg acggcttttg gctggataac agcaagaaca tggcgggtgg ccaaaaagag 60
<210> 75
<211> 60
<212> DNA
<213> modified bacterium
<400> 75
attccggaat tcgtggcgat gctgcgtgac attgatccga gctttgcgat cggtgttaac 60
<210> 76
<211> 60
<212> DNA
<213> modified bacterium
<400> 76
tacgaaaccc actatttcga ggacgaagat ggcaactacc tgaaggttgc gagcgacagc 60
<210> 77
<211> 60
<212> DNA
<213> modified bacterium
<400> 77
atcgacgata acgacgatcg tgaatacaag atcattaaac acgtggttac caacgagtat 60
<210> 78
<211> 60
<212> DNA
<213> modified bacterium
<400> 78
atggatttca ccaacggtca tgtgaccccg atgggtcaag gtgctccgcc gaacagctgg 60
<210> 79
<211> 60
<212> DNA
<213> modified bacterium
<400> 79
ggctacgagg aatataccat tccgcacatg atcgaaaaac cgtgggacag cgttgatggt 60
<210> 80
<211> 60
<212> DNA
<213> modified bacterium
<400> 80
aaccactatg cgctgatgca cggctggttc ccgattcgtt ttagctggag cggtagcggc 60
<210> 81
<211> 60
<212> DNA
<213> modified bacterium
<400> 81
gcggagctga tgttcgaaac cgaacaggcg taccgttttg ttcgtaccat taccgatggt 60
<210> 82
<211> 60
<212> DNA
<213> modified bacterium
<400> 82
ggcgcggcga tgacctggag caccacccaa aagaaaggtt atatgagcgc ggacgaaatg 60
<210> 83
<211> 60
<212> DNA
<213> modified bacterium
<400> 83
gatatcatga ttgagatcaa caaccgtatg acccaagcgc cgaagctgga ttacgaggcg 60
<210> 84
<211> 45
<212> DNA
<213> modified bacterium
<400> 84
tatgaacgtc cggagggtgc gtacctggtt ggcgagatcg aataa 45
<210> 85
<211> 60
<212> PRT
<213> amino acid
<400> 85
Met Leu Ile Ser Val Thr Val Leu Val Gly Cys Gly Ser Ser Ser Asp
1 5 10 15
Glu Val Glu Ser Thr Asn Thr Thr Asp Ser Tyr Asn Ile Asn Asn Thr
20 25 30
Glu Ala Thr Gly Ile Glu Tyr Asn Ala Ser Trp Met Ala Gly Thr Trp
35 40 45
Gly Ile Thr Gln Arg Val Asp Gly Gly Tyr Lys Leu
50 55 60
<210> 86
<211> 60
<212> PRT
<213> amino acid
<400> 86
Asp Asn Ser Ala Asp Ser Ser Asn Trp Gln Ala Gly Ala Glu Glu Ile
1 5 10 15
Val Thr Asn Ile Pro Ala Ala Glu Tyr Val Ile Thr Ser Phe Thr His
20 25 30
Pro Ala His Gly His Leu Phe Thr Leu Arg Thr Asn Asn Asn Val Asp
35 40 45
Val Ser Ala Ile His Pro Asp Met Tyr Pro Thr Leu
50 55 60
<210> 87
<211> 60
<212> PRT
<213> amino acid
<400> 87
Glu Asn Glu Lys Ile Ile Leu Asp Val Ile Asn Ile Tyr Arg Ala Ala
1 5 10 15
Gly Lys Lys Val Ile Leu Tyr Leu Asn Ser Ala Asp Pro Ser Met Ala
20 25 30
Glu Glu Arg Gly Asp Thr Asp Ile Gln Ala Ala Trp Asp Glu Tyr Tyr
35 40 45
Ile Asn Glu Trp Asp Gly Asp Glu Ala Ala Ala Trp
50 55 60
<210> 88
<211> 60
<212> PRT
<213> amino acid
<400> 88
Arg Asn Leu Ala Arg Gly Tyr Val Glu Arg Phe Asp Gly Leu Val Asp
1 5 10 15
Gly Tyr Trp Ile Leu Asp Asn Ser Arg Asn Leu Pro Gly Glu Val Ser
20 25 30
Asp Phe Val Ala Met Leu Arg Ser Val Asp Pro Glu Leu Thr Ile Ala
35 40 45
Val Asn Tyr Asp His Tyr Phe Thr Asp Asp Asn Gly
50 55 60
<210> 89
<211> 60
<212> PRT
<213> amino acid
<400> 89
Glu Tyr Leu Tyr Val Asp Ser Asp Gly Leu Asp Asp Glu Asp Glu Ser
1 5 10 15
Asp Tyr Lys Ile Val Lys His Val Val Thr Asn Glu Tyr Met Asp Phe
20 25 30
Thr Asn Gly His Val Thr Pro Leu Gly Arg Gly Ala Pro Pro Asn Ser
35 40 45
Trp Ala Tyr Glu Glu Asp Thr Ile Pro Asp Met Ile
50 55 60
<210> 90
<211> 60
<212> PRT
<213> amino acid
<400> 90
Glu Val Pro Trp Glu Thr Tyr Asp Gly Ser Lys Tyr Ala Leu Lys His
1 5 10 15
Gly Trp Phe Pro Ile Arg Asn Ser Trp Ser Gly Ser Lys Ala Glu Leu
20 25 30
Met Phe Asp Val Glu Gln Ala Tyr Arg Phe Val Arg Thr Val Thr Asp
35 40 45
Gly Gly Ala Ala Met Thr Trp Ser Thr Thr Gln Asp
50 55 60
<210> 91
<211> 43
<212> PRT
<213> amino acid
<400> 91
Asn Gly Tyr Met Thr Ala Asp Glu Met Ser Ile Met Ile Glu Ile Ser
1 5 10 15
Asn Arg Met Thr Gln Thr Pro Lys Pro Asp Tyr Ser Val Tyr Glu Arg
20 25 30
Pro Lys Gly Ala Tyr Leu Val Ser Glu Ile Glu
35 40
<210> 92
<211> 60
<212> PRT
<213> amino acid
<400> 92
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Gly Ser Ser Ser Asp Glu Val Glu Ser Thr Asn
20 25 30
Thr Thr Asp Ser Tyr Asn Ile Asn Asn Thr Glu Ala Thr Gly Ile Glu
35 40 45
Tyr Asn Ala Ser Trp Met Ala Gly Thr Trp Gly Ile
50 55 60
<210> 93
<211> 60
<212> PRT
<213> amino acid
<400> 93
Thr Gln Arg Val Asp Gly Gly Tyr Lys Leu Asp Asn Ser Ala Asp Ser
1 5 10 15
Ser Asn Trp Gln Ala Gly Ala Glu Glu Ile Val Thr Asn Ile Pro Ala
20 25 30
Ala Glu Tyr Val Ile Thr Ser Phe Thr His Pro Ala His Gly His Leu
35 40 45
Phe Thr Leu Arg Thr Asn Asn Asn Val Asp Val Ser
50 55 60
<210> 94
<211> 60
<212> PRT
<213> amino acid
<400> 94
Ala Ile His Pro Asp Met Val Pro Thr Leu Glu Asn Glu Lys Ile Ile
1 5 10 15
Leu Asp Val Ile Asn Ile Tyr Arg Ala Ala Gly Lys Lys Val Ile Leu
20 25 30
Tyr Leu Asn Ser Ala Gly Pro Ser Met Ala Glu Glu Arg Gly Asp Thr
35 40 45
Asp Ile Gln Ala Ala Trp Asp Glu Tyr Tyr Ile Asn
50 55 60
<210> 95
<211> 60
<212> PRT
<213> amino acid
<400> 95
Glu Trp Asp Gly Asp Glu Ala Ala Ala Trp Arg Asn Leu Ala Arg Gly
1 5 10 15
Tyr Val Glu Arg Phe Asp Gly Leu Val Asp Gly Tyr Trp Leu Asp Asn
20 25 30
Ser Arg Asn Leu Pro Gly Glu Val Ser Asp Phe Val Ala Met Leu Arg
35 40 45
Ser Val Asp Pro Glu Leu Thr Ile Ala Val Asn Tyr
50 55 60
<210> 96
<211> 60
<212> PRT
<213> amino acid
<400> 96
Asp Gln His Tyr Phe Thr Asp Asp Asn Gly Glu Tyr Leu Tyr Val Asp
1 5 10 15
Ser Asp Gly Leu Asp Asp Glu Asp Glu Ser Asp Tyr Lys Ile Val Lys
20 25 30
His Val Val Thr Asn Glu Tyr Met Asp Phe Thr Asn Gly His Val Thr
35 40 45
Pro Leu Gly Arg Gly Ala Pro Pro Asn Ser Trp Ala
50 55 60
<210> 97
<211> 60
<212> PRT
<213> amino acid
<400> 97
Tyr Glu Glu Tyr Thr Ile Pro Asp Met Ile Glu Val Pro Trp Glu Thr
1 5 10 15
Tyr Asp Gly Ser Lys Tyr Ala Leu Lys His Gly Trp Phe Pro Ile Arg
20 25 30
Asn Ser Trp Ser Gly Ser Lys Ala Glu Leu Met Phe Asp Val Glu Gln
35 40 45
Ala Tyr Arg Phe Val Arg Thr Val Thr Asp Gly Gly
50 55 60
<210> 98
<211> 53
<212> PRT
<213> amino acid
<400> 98
Ala Ala Met Thr Trp Ser Thr Thr Gln Asp Asn Gly Tyr Met Thr Ala
1 5 10 15
Asp Glu Met Ser Ile Met Ile Glu Ile Ser Asn Arg Met Thr Gln Thr
20 25 30
Pro Lys Pro Asp Tyr Ser Val Tyr Glu Arg Pro Lys Gly Ala Tyr Leu
35 40 45
Val Ser Glu Ile Glu
50
<210> 99
<211> 60
<212> PRT
<213> amino acid
<400> 99
Met Leu Ile Gly Cys Gly Gly Ser Ser Ala Asn Gln Thr Gln Ser Thr
1 5 10 15
Ser Asp Gln Asp Ser Ser Glu Val Asn Glu Thr Val Ser Leu Asp Ser
20 25 30
Glu Tyr Thr Ala Asn Trp Met Ala Gly Ala Trp Gly Ile Thr Gln Arg
35 40 45
Val Asp Gly Gly Tyr Lys Leu Asp Ala Ser Val Glu
50 55 60
<210> 100
<211> 60
<212> PRT
<213> amino acid
<400> 100
Thr Gly Lys Tyr Asp Trp Val Ala Gly Ala Glu Glu Ile Val Glu Asn
1 5 10 15
Ile Pro Ser Ala Gly Tyr Val Ile Thr Ser Phe Thr His Pro Ala His
20 25 30
Gly Phe Leu Tyr Thr Leu Arg Asp Asn Glu Asn Val Asp Val Ala Ala
35 40 45
Ile His Pro Asp Met Val Pro Ser Leu Glu Asn Glu
50 55 60
<210> 101
<211> 60
<212> PRT
<213> amino acid
<400> 101
Lys Ile Ile Phe Asp Val Ile Asn Val Tyr Lys Ser Ala Gly Lys Lys
1 5 10 15
Val Leu Leu Tyr Leu Asn Thr Ala Gly Pro Thr His Ala Ala Asp Arg
20 25 30
Asn Ser Pro Glu Ile Gln Asp Ala Trp Asp Asp Tyr Val Asn Thr Asn
35 40 45
Trp Asn Gly Asp His Gly Ala Ala Trp Arg Asn Leu
50 55 60
<210> 102
<211> 60
<212> PRT
<213> amino acid
<400> 102
Val Glu Gly Tyr Ala Lys Arg Phe Lys Gly Leu Val Asp Gly Phe Trp
1 5 10 15
Leu Asp Asn Ser Lys Asn Met Ala Gly Gly Gln Lys Glu Ile Pro Glu
20 25 30
Phe Val Ala Met Leu Arg Asp Ile Asp Pro Ser Phe Ala Ile Gly Val
35 40 45
Asn Tyr Glu Thr His Tyr Phe Glu Asp Glu Asp Gly
50 55 60
<210> 103
<211> 60
<212> PRT
<213> amino acid
<400> 103
Asn Tyr Leu Lys Val Ala Ser Asp Ser Ile Asp Asp Asn Asp Asp Arg
1 5 10 15
Glu Tyr Lys Ile Ile Lys His Val Val Thr Asn Glu Tyr Met Asp Phe
20 25 30
Thr Asn Gly His Val Thr Pro Met Gly Gln Gly Ala Pro Pro Asn Ser
35 40 45
Trp Gly Tyr Glu Glu Tyr Thr Ile Pro His Met Ile
50 55 60
<210> 104
<211> 60
<212> PRT
<213> amino acid
<400> 104
Glu Lys Pro Trp Asp Ser Val Asp Gly Asn His Tyr Ala Leu Met His
1 5 10 15
Gly Trp Phe Pro Ile Arg Phe Ser Trp Ser Gly Ser Gly Ala Glu Leu
20 25 30
Met Phe Glu Thr Glu Gln Ala Tyr Arg Phe Val Arg Thr Ile Thr Asp
35 40 45
Gly Gly Ala Ala Met Thr Trp Ser Thr Thr Gln Lys
50 55 60
<210> 105
<211> 43
<212> PRT
<213> amino acid
<400> 105
Lys Gly Tyr Met Ser Ala Asp Glu Met Asp Ile Met Ile Glu Ile Asn
1 5 10 15
Asn Arg Met Thr Gln Ala Pro Lys Leu Asp Tyr Glu Ala Tyr Glu Arg
20 25 30
Pro Glu Gly Ala Tyr Leu Val Gly Glu Ile Glu
35 40
<210> 106
<211> 60
<212> PRT
<213> amino acid
<400> 106
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Asn Gln Thr Gln Ser Thr Ser Asp Gln Asp Ser
20 25 30
Ser Glu Val Asn Glu Thr Val Ser Leu Asp Ser Glu Tyr Thr Ala Asn
35 40 45
Trp Met Ala Gly Ala Trp Gly Ile Thr Gln Arg Val
50 55 60
<210> 107
<211> 60
<212> PRT
<213> amino acid
<400> 107
Asp Gly Gly Tyr Lys Leu Asp Ala Ser Val Glu Thr Gly Lys Tyr Asp
1 5 10 15
Trp Val Ala Gly Ala Glu Glu Ile Val Glu Asn Ile Pro Ser Ala Gly
20 25 30
Tyr Val Ile Thr Ser Phe Thr His Pro Ala His Gly Phe Leu Tyr Thr
35 40 45
Leu Arg Asp Asn Glu Asn Val Asp Val Ala Ala Ile
50 55 60
<210> 108
<211> 60
<212> PRT
<213> amino acid
<400> 108
His Pro Asp Met Val Pro Ser Leu Glu Asn Glu Lys Ile Ile Phe Asp
1 5 10 15
Val Ile Asn Val Tyr Lys Ser Ala Gly Lys Lys Val Leu Leu Tyr Leu
20 25 30
Asn Thr Ala Gly Pro Thr His Ala Ala Asp Arg Asn Ser Pro Glu Ile
35 40 45
Gln Asp Ala Trp Asp Asp Tyr Val Asn Thr Asn Trp
50 55 60
<210> 109
<211> 60
<212> PRT
<213> amino acid
<400> 109
Asn Gly Asp His Gly Ala Ala Trp Arg Asn Leu Val Glu Gly Tyr Ala
1 5 10 15
Lys Arg Phe Lys Gly Leu Val Asp Gly Phe Trp Leu Asp Asn Ser Lys
20 25 30
Asn Met Ala Gly Gly Gln Lys Glu Ile Pro Glu Phe Val Ala Met Leu
35 40 45
Arg Asp Ile Asp Pro Ser Phe Ala Ile Gly Val Asn
50 55 60
<210> 110
<211> 60
<212> PRT
<213> amino acid
<400> 110
Tyr Glu Thr His Tyr Phe Glu Asp Glu Asp Gly Asn Tyr Leu Lys Val
1 5 10 15
Ala Ser Asp Ser Ile Asp Asp Asn Asp Asp Arg Glu Tyr Lys Ile Ile
20 25 30
Lys His Val Val Thr Asn Glu Tyr Met Asp Phe Thr Asn Gly His Val
35 40 45
Thr Pro Met Gly Gln Gly Ala Pro Pro Asn Ser Trp
50 55 60
<210> 111
<211> 60
<212> PRT
<213> amino acid
<400> 111
Gly Tyr Glu Glu Tyr Thr Ile Pro His Met Ile Glu Lys Pro Trp Asp
1 5 10 15
Ser Val Asp Gly Asn His Tyr Ala Leu Met His Gly Trp Phe Pro Ile
20 25 30
Arg Phe Ser Trp Ser Gly Ser Gly Ala Glu Leu Met Phe Glu Thr Glu
35 40 45
Gln Ala Tyr Arg Phe Val Arg Thr Ile Thr Asp Gly
50 55 60
<210> 112
<211> 54
<212> PRT
<213> amino acid
<400> 112
Gly Ala Ala Met Thr Trp Ser Thr Thr Gln Lys Lys Gly Tyr Met Ser
1 5 10 15
Ala Asp Glu Met Asp Ile Met Ile Glu Ile Asn Asn Arg Met Thr Gln
20 25 30
Ala Pro Lys Leu Asp Tyr Glu Ala Tyr Glu Arg Pro Glu Gly Ala Tyr
35 40 45
Leu Val Gly Glu Ile Glu
50
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