WO2012142326A1 - A method of producing 5-hydroxypyridine-2-carboxylic acid from alginate - Google Patents

Abstract

The present application provides methods for converting alginate into 5-hydroxypyridine- 2-carboxylic acid (5-HPA) using an alginate lyase and a recombinant oligoalginate lyase. The alginate lyase breaks down alginate into oligoalginate. The oligoalginate lyase breaks down the oligoalginate into monomers such as 4-deoxy-L-erythro-hexoseulose uronate (DEHU), which serves as a precursor for conversion into 5-HPA. The addition of ammonia or an ammonium ion further drives the conversion of DEHU into 5-HPA.

Description

A METHOD OF PRODUCING 5-HYDROXYPYRIDINE-2-CARBOXYLIC ACID FROM

ALGINATE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/474,709 filed April 12, 2011, and U.S. Provisional Patent Application No. 61/535,909 filed September 16, 2011, each of which are incorporated herein by reference in their entireties.

BACKGROUND

1. Field

[0002] The present disclosure relates generally to a method of producing 5-hydroxypyridine- 2-carboxylic acid (5-HPA). In particular, the present disclosure relates to a method of producing 5-HPA from enzymatically-degraded alginate in the presence of ammonia or an ammonium ion.

2. Description of Related Art

[0003] 5-hydroxypyridine-2-carboxylic acid (5-HPA) may be useful as a precursor for pesticides, plant growth enhancers, pharmaceuticals, surfactants, and polymers (e.g., materials, plastics, and fabrics).

[0004] Several synthetic methods are available for making 5-HPA. For example, 5-HPA can be made from 3-hydroxypyridine in the presence of carbon dioxide at a temperature above 150°C and a pressure above 80 kg/cm². See J. Org. Chem., 1954, Vol. 19, p.510. 5-HPA can also be made from renewable sources, such as alginate. JP6256310 describes a method for producing 5-HPA from alginate using Alteromonas sp., in which the bacterium incorporates alginate into its cells to produce 5-HPA. Bacterial systems such as Alteromonas sp., however, often use alginate in its metabolic pathways for growth that compete with the production of 5- HPA. See e.g., Carbohydr. Res., 1997, 304(1): 69-76.

[0005] Thus, what is needed in the art is an improved method of producing 5-HPA from renewable resources, such as alginate.

BRIEF SUMMARY

[0006] The present disclosure addresses this need by providing a method to produce 5-HPA by contacting alginate directly with an alginate lyase (AL) and/or an oligoalginate lyase (OAL). In one aspect, the present disclosure provides a method for producing 5-hydroxypyridine-2- carboxylic acid (5-HPA) by: a) providing oligoalginate; b) providing an isolated oligoalginate lyase; c) contacting the oligoalginate with the isolated oligoalginate lyase to form 4-deoxy-L- erythro-5-hexoseulose uronate (DEHU); and d) converting at least a portion of the DEHU to 5- HPA. In some embodiments, the method may be performed at a pH between about 6.0 and about 7.6. In other embodiments, the method may be performed at a pH between about 7.2 and about 7.6. In one embodiment, the method may be performed at a pH of about 7.5. In certain embodiments that may be combined with the preceding embodiments, the method may be performed at a temperature between about 18°C and about 50°C.

[0007] In certain embodiments that may be combined with the preceding embodiments, the oligoalginate is obtained from sodium alginate. In other embodiments, the oligoalginate is obtained from a member of the species selected from the group consisting of Saccharina, Macrocystis, Undaria, and Ascophyllum. In yet other embodiments, the oligoalginate is obtained from Saccharina japonica, Macrocystis pyrifera, Saccharina latissima, Saccharina digitata, Undaria pinnatifida, Ascophyllum nodosum, Saccharina angustata, or Sargassum muticum. In one embodiment, the oligoalginate is obtained from Saccharina japonica or Macrocystis pyrifera. In another embodiment, the oligoalginate is obtained from Undaria pinnatifida, Saccharina latissima, or Ascophyllum nodosum. The oligoalginate may also be obtained from a combination of sources. In certain embodiments, the oligoalginate includes one or more alginate dimers, trimers, and tetramers.

[0008] In certain embodiments that may be combined with the preceding embodiments, the oligoalginate lyase is isolated from a recombinant source. In some embodiments, the

oligoalginate lyase is isolated from E. coli or yeast.

[0009] In certain embodiments that may be combined with the preceding embodiments, the method further includes adding ammonia or an ammonium salt, wherein the addition converts at least a portion of the DEHU into the 5-HPA. In some embodiments, the ammonium salt is ammonium chloride, ammonium phosphate, ammonium acetate, ammonium nitrate, ammonium sulfate, or a combination thereof. In one embodiment, the ammonium salt is ammonium chloride or ammonium hydroxide. In certain embodiments that may be combined with any of the preceding embodiments, the concentration of the ammonia or ammonium salt is between 100 mM and 600 mM. In other embodiments, the concentration of the ammonia or ammonium salt is between lOOmM and 150 mM. In yet other embodiments, the method further includes adding a phosphate salt, in addition to the ammonia or the ammonium salt, to convert at least a portion of the DEHU into the 5-HPA.

[0010] In some embodiments that may be combined with the preceding embodiments, the method further includes isolating the 5-HPA. In some embodiments, the isolating of the 5-HPA includes precipitating the 5-HPA at a pH of 4.5 or below. In other embodiments, the method further includes purifying the isolated 5-HPA by precipitation. In certain embodiments, the precipitation is performed at a pH of 4.5 or below. In yet other embodiments that may be combined with the preceding embodiments, the method employs a fed-batch process.

[0011] In another aspect, the present disclosure provides a method for producing 5- hydroxypyridine-2-carboxylic acid (5-HPA) by: a) providing alginate; b) providing an isolated alginate lyase; c) providing an isolated oligoalginate lyase; d) contacting the alginate with the isolated alginate lyase to form oligoalginate; e) contacting the oligoalginate with the isolated oligoalginate lyase to form 4-deoxy-L-erythro-5-hexoseulose uronate (DEHU); and f) converting at least a portion of the DEHU to 5-HPA.

[0012] In some embodiments, the contacting of the alginate with the isolated alginate lyase to form oligoalginate and the contacting of the oligoalginate with the isolated oligoalginate lyase to form DEHU is performed together in a one -pot reaction. In one variation, the method includes contacting the alginate with the isolated alginate lyase and the isolated oligoalginate lyase simultaneously to form DEHU, which can be converted into 5-HPA. In other

embodiments, the oligoalginate formed from the alginate is isolated before contacting the oligoalginate with the isolated oligoalginate lyase to form DEHU.

[0013] In some embodiments, the method may be performed at a pH between about 4.0 and about 9.0, between about 6.0 and about 8.5, between about 6.5 and about 8.3, between about 6.0 and 8.0, between about 6.0 and about 7.6, or between about 7.2 and about 7.6. In one embodiment, the method may be performed at a pH of about 7.5. In certain embodiments that may be combined with the preceding embodiments, the method may be performed at a temperature between about 18°C and about 50°C.

[0014] In certain embodiments that may be combined with the preceding embodiments, the alginate has a concentration of about 1-100 g/L. In other embodiments, the alginate has a concentration of about 5-50 g/L. In yet other embodiments, the alginate has a concentration of about 5-20 g/L. [0015] In one embodiment that may be combined with the preceding embodiments, the alginate is sodium alginate. In other embodiments, the oligoalginate is obtained from a member of the species selected from the group consisting of Saccharina, Macrocystis, Undaria, and

Ascophyllum. In yet other embodiments, the oligoalginate is obtained from Saccharina japonica,

Macrocystis pyrifera, Saccharina latissima, Saccharina digitata, Undaria pinnatifida,

Ascophyllum nodosum, Saccharina angustata, or Sargassum muticum. In one embodiment, the oligoalginate is obtained from Saccharina japonica or Macrocystis pyrifera. In another embodiment, the oligoalginate is obtained from Undaria pinnatifida, Saccharina latissima, or

Ascophyllum nodosum. The oligoalginate may also be obtained from a combination of sources.

In certain embodiments, the oligoalginate includes one or more alginate dimers, trimers, and tetramers.

[0016] In certain embodiments that may be combined with the preceding embodiments, the alginate lyase is isolated from a recombinant source. In certain embodiments that may be combined with the preceding embodiments, the oligoalginate lyase is isolated from a

recombinant source. In some embodiments, the oligoalginate lyase is isolated from E. coli or yeast.

[0017] In certain embodiments that may be combined with the preceding embodiments, the method further includes adding ammonia or an ammonium salt, wherein the addition converts at least a portion of the DEHU into the 5-HPA. In some embodiments, the ammonium salt is ammonium chloride, ammonium phosphate, ammonium acetate, ammonium nitrate, ammonium sulfate, or a combination thereof. In one embodiment, the ammonium salt is ammonium chloride or ammonium hydroxide. In certain embodiments that may be combined with any of the preceding embodiments, the concentration of the ammonia or ammonium salt is between 100 mM and 600 mM. In other embodiments, the concentration of the ammonia or ammonium salt is between lOOmM and 150 mM. In yet other embodiments, the ammonia or ammonium salt is added at a ratio of 0.32-6.7 mole relative to 1 mole of DEHU. In yet other embodiments, the method further includes adding a phosphate salt, in addition to the ammonia or ammonium salt, to convert at least a portion of the DEHU into the 5-HPA.

[0018] In some embodiments that may be combined with the preceding embodiments, the method further includes isolating the 5-HPA. In some embodiments, the isolating of the 5-HPA includes precipitating the 5-HPA at a pH of 4.5 or below. In other embodiments, the method further includes purifying the isolated 5-HPA by precipitation. In certain embodiments, the precipitation is performed at a pH of 4.5 or below. In yet other embodiments that may be combined with the preceding embodiments, the method employs fed-batch process. In certain embodiments that may be combined with the preceding embodiments, the method yields at least 32% of the theoretical maximum of 5-HPA that may be produced from the alginate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present application can be best understood by reference to the following description taken in conjunction with the accompanying drawing figures, in which like parts may be referred to by like numerals:

[0020] FIG. 1 is an exemplary reaction depicting the conversion of alginate into 5-HPA using an alginate lyase and a recombinant oligoalginate lyase in the presence of ammonia;

[0021] FIG. 2 depicts the assignment of peaks observed from the 1H NMR of a sample obtained from a reaction mixture containing 5-HPA;

[0022] FIG. 3 is an overlay of HPLC-MS chromatograms of an oligoalginate lyase-degraded sample in which the only source of ammonium ion comes from the M9 salts in the sigma alginate lyase (10 mg/mL stock solution);

[0023] FIG. 4 is a HPLC-MS chromatogram of an oligoalginate lyase-degraded sample in which ammonium sulfate was added;

[0024] FIG. 5 is an overlay of two HPLC-UV chromatograms of a Bal 791 (alginolytic fungi) culture-free supernatant sample: (1) the initial time period represents the time

immediately after ammonium sulfate was added, and (2) the end time period represents 5 hours after the ammonium sulfate was added;

[0025] FIG. 6 is a graph depicting the standard curves for a sample obtained from a reaction mixture containing 5-HPA ("Alg-"); a sample of 5-HPA obtained from DATO Chemicals Company, Limited (China) ("DATO"); and a sample of 5-HPA carboxylate sodium salt obtained from Indofine Chemical Company (New Jersey, USA) ("ICC");

[0026] FIG. 7 is a graph illustrating the production of 5-HPA in the reaction as a function of time at several temperatures, i.e., 25°C ("D25"), 30°C ("D30"), 37°C ("D37"), and 45°C ("D45"); [0027] FIG. 8 is a graph illustrating the production of 5-HPA in the reaction as a function of time at several pH values ranging from pH 6 to 7.6, and at pH 7 with additional 20 mM of ammonium chloride;

[0028] FIG. 9 is a graph illustrating the percent yield of 5-HPA from alginate as a function of different pH values, ranging from 4.67 to 8.26, under the following conditions: "Series A" = 100 mM phosphate, 0.4% alginate, lx AL/OAL loading; "Series B" = 100 mM phosphate, 1.0% alginate, lx AL/OAL loading; "Series C" = 50 mM phosphate, 0.4% alginate, lx AL/OAL loading; "Series D" = 50 mM phosphate, 1.0% alginate, 5x AL/OAL loading; "Series E" = 10 mM phosphate, 0.4% alginate, 5x AL/OAL loading; and "Series F" = 10 mM phosphate, 1.0% alginate, 5x AL/OAL loading;

[0029] FIG. 10A is a graph illustrating the percent yield of 5-HPA from DEHU as a function of pH values, ranging from 4.45 to 8.07, under the conditions described above for Series A-F;

[0030] FIG. 10B is a graph illustrating the percent yield of 5-HPA from DEHU as a function of pH values, ranging from 8 to 11, under the conditions described above for Series A, B, E and F;

[0031] FIG. 11A is a graph illustrating the amount of 5-HPA produced in the reaction as a function of time at (1) pH 7, and (2) pH 7 with addition of 20 mM ammonium chloride;

[0032] FIG. 11B is a graph comparing the amount of 5-HPA produced in a reaction with 4.5 mM equivalent of DEHU as a function of ammonium chloride concentration, ranging from 0-30 mM (i.e., 0-6.7 mole equivalent of ammonium chloride to 1 mole of DEHU);

[0033] FIG. 12 is a graph depicting the standard curves for a sample of 5-HPA produced from four different alginate sources purchased from Sigma ("Alg A2158", "Alg A2033", "Alg A0682", and "Alg 180947"), and a sample of 5-HPA obtained from DATO Chemicals

Company, Limited (China) ("DATO 5HPA");

[0034] FIG. 13 is a graph depicting 5-HPA formation using different alginate lyases: Sigma alginate lyase ("Sigma"), Pseudoalteromonas sp. SM0524 alginate lyase ("SM0524"), and Sphingomonas sp. Al alginate lyase ("Al-1"); [0035] FIG. 14 is a graph depicting 5-HPA formation in the presence of different oligoalginate lyases (Vibrio splendidus 12B01oligoalginate lyase (V12B01_24239) and BAL stock # 966 oligoalginate lyase) and in the absence of oligoalginate lyase;

[0036] FIGS. 15A and 15B are graphs illustrating the amount of 5-HPA produced (g/L) and the percent yield of 5-HPA produced, respectively, in the reaction as a function of time at different initial ammonium chloride concentrations (110 mM and 555 mM) and pH (6.5 and 7.5);

[0037] FIG. 16 is a graph illustrating the amount of 5-HPA produced in the reaction as a function time at different temperatures (18-22°C and 30°C);

[0038] FIG. 17 is a graph illustrating the amount of 5-HPA produced in the reaction from Saccharina japonica and Macrocystis pyrifera as a function of time;

[0039] FIG. 18 is a graph illustrating the amount of 5-HPA produced in a fed-batch process at 30°C as a function of time;

[0040] FIGS. 19A and 19B are graphs illustrating the yield of 5-HPA in the supernatant and the yield of 5-HPA in the supernatant and precipitated pellet, respectively, as a function of different alginate concentrations, ranging from 0.625 g/L to 20.000 g/L, at (1) "G odd" = 100 mM phosphate, 188 mM ammonium chloride; (2) "G even" = 100 mM phosphate, 656 mM ammonium chloride; (3) "H odd" = 50 mM phosphate, 188 mM ammonium chloride; (4) "H even" = 50 mM phosphate, 656 mM ammonium chloride; (5) "I odd" = 10 mM phosphate, 188 mM ammonium chloride; (6) "I even" = 10 mM phosphate, 188 mM ammonium chloride, (7) "J odd" = 188 mM ammonium chloride; (8) "J even" = 656 mM ammonium chloride.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0042] The following description relates to methods for synthesizing 5-hydroxypyridine-2- carboxylic acid (5-HPA). In one method described herein, 5-HPA is synthesized directly from alginate, a sugar polymer typically found in seaweed. Alginate is first degraded by endo- and exo-type alginate lyases to form 4-deoxy-L-erythro-hexoseulose uronate (DEHU). DEHU is then converted into 5-HPA in the presence of ammonia or an ammonium ion in an aqueous solution.

[0043] With reference to FIG. 1, reaction 100 is an exemplary embodiment that depicts the conversion of alginate into oligoalginate and subsequently into 5-HPA using two enzymes: (1) an alginate lyase, and (2) an oligoalginate lyase. The alginate and oligoalginate used in the methods described herein are contacted with alginate lyase and oligoalginate lyase outside of a cell. As used herein, "isolated alginate lyase" and "isolated oligoalginate lyase" refers to lyases that are not within the cell. In some embodiments, the isolated alginate lyase or isolated oligoalginate lyase may be partially purified, purified or be part of a cellular extract. In some embodiments, the isolated alginate lyase or isolated oligoalginate lyase may be excreted or secreted by a cell. The isolated alginate lyase or isolated oligoalginate lyase may be

recombinant.

[0044] Isolated alginate lyase 104 breaks down alginate 102 into oligoalginate 106, which may include, for example, alginate dimers, trimers, and tetramers. It should be understood that oligoalginate 106 may include other oligomers of varying lengths depending on the lyases used and the reaction conditions. Isolated oligoalginate lyase 108 is added to the reaction mixture to break down oligoalginate 106 into DEHU 110, which is subsequently converted into 5-HPA 114 upon addition of ammonia 112 to the reaction mixture. In other exemplary embodiments, DEHU can be converted into 5-HPA in the presence of an ammonium ion in an aqueous solution. In yet other exemplary embodiments, DEHU can be converted into 5-HPA in the presence of ammonia or an ammonium ion, and a phosphate ion.

[0045] It should be noted that one or more steps may be combined, omitted or added from reaction 100. For example, in other embodiments, oligoalginate 106 formed from alginate 102 is isolated before contacting the oligoalginate with isolated oligoalginate lyase 108 to form DEHU 110, which can be converted into 5-HPA 114.

[0046] The present description also provides a method for synthesizing 5-hydroxypyridine-2- carboxylic acid (5-HPA) directly from oligoalginate made up of oligoalginates using an isolated oligoalginate lyase and the addition of ammonia or an ammonium ion to the reaction mixture. In some embodiments, the oligoalginate may be obtained from alginate, or from any other methods currently known in the art. Alginate

[0047] Alginate is a linear copolymer with homopolymeric blocks of (l-4)-linked β-D- mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. Alginate monomers may appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks), alternating M- and G-residues (MG-blocks), or randomly organized blocks.

[0048] The alginate used in reaction 100 may be, in some embodiments, in the form of sodium alginate. In one embodiment, the alginate is polymeric sodium alginate.

[0049] Moreover, the alginate used in reaction 100 may be obtained from any source known to one skilled in the art. Suitable sources of alginate may include kelp, giant kelp, sargasso, macroalgae (or seaweed), brown algae, and bacterial culture (Pseudomonas sp.). For example, in some embodiments, the alginate may be obtained from Saccharina japonica, Macrocystis pyrifera, Saccharina latissima, Saccharina digitata, Undaria pinnatijida, Ascophyllum nodosum, Saccharina angustata, or Sargassum muticum.

[0050] The alginate concentration used in the reaction may affect reaction rate and yield of producing 5-HPA. In certain embodiments, the alginate used in the reaction has a concentration of about 1-100 g/L. In other embodiments, the alginate used in the reaction has a concentration of about 1-50 g/L, 1-20 g/L, 1-15 g/L, 1-10 g/L, 5-50 g/L, 10-50 g/L, 5-25 g/L, or 10-25 g/L.

Forming Oligoalginate

[0051] Alginate may be broken down into smaller subunits known as oligoalginates by alginate lyases. It should be understood that oligoalginates formed by breaking down alginate may have varying lengths depending on the enzymes used and the reaction conditions. The oligoalginate may have two to several hundred monomer units. For example, in some embodiments, the oligoalginate may include alginate dimers (e.g., disaccharides), trimers (e.g., trisaccharides), tetramers (e.g., tetrasaccharides), pentamers (e.g., pentasaccharides), hexamers (e.g., hexasaccharides) and heptamers (e.g., heptasaccharides).

[0052] The family of enzymes used in reaction 100 is mainly classified into two distinctive subfamilies: endo-acting alginate lyases, and exo-acting alginate lyases. Alginate lyase 104 may be any endo-acting alginate lyases that can depolymerize the alginate into oligosaccharides, which may include, for example, disaccharides, trisaccharides, and tetrasaccharides. [0053] Endo-acting alginate lyases typically cleave internal glycosidic bonds. Examples may include mannuronate lyases (EC 4.2.2.3). Endo-acting alginate lyases are further classified based on their catalytic specificity: M-specific and G-specific alginate lyases. These endo- acting alginate lyases randomly cleave alginate via a β-elimination mechanism to depolymerize the alginate into oligoalginate, which may include dimers (e.g., disaccharides), trimers (e.g., trisaccharides), and tetramers (e.g., tetrasaccharides).

[0054] Suitable alginate lyases used to break down alginate polymer into oligomers (e.g., dimers, trimers, tetramers) may include, for example, polymannuronate lyases, polyguluronate lyases, polygalacturonate lyases, and hyaluronan lyases. Alginate lyases may be isolated from various sources, including marine algae, mollusks, and wide varieties of microbes such as genus Pseudomonas, Vibrio, and Sphingomonas. See e.g., T.Y. Wong et al, Alginate Lyase: Review of Major Sources and Enzyme Characteristics, Structure-Function Analysis, Biological Roles, and Applications, ANNU. REV. MICROBIOL. 2000. 54: 289-340.

[0055] For example, a bacterium, Sphingomonas sp. strain Al can incorporate alginate into the cells, and depolymerize the incorporated alginate by three types of cytoplasmic alginate lyases: Al-I (66 kDa), Al-II (25 kDa), and Al-III (40 kDa). Al-I is self-spliced to produce Al-II and Al-III. These three lyases endolytically cleave the alginate polymer by β-elimination reaction. Al-I may have high activity for both M and G residues, whereas Al-II may be more specific to G-residues and Al-III may be more specific to M-residues. In addition to these three alginate lyases, two additional endo-acting alginate lyases (i.e., Al-ΙΓ and Al-IV) and one exo- acting alginate lyase (i.e., Al-IV) have been isolated from Sphingomonas sp. strain Al. Al-IV has molecular weight of about 85 kDa, and catalyzes exolytic depolymerization of alginate. Although both Al-ΙΓ and Al-IV are functional homologues of Al-II and Al-IV, ΑΙ-ΙΓ has endolytic activity and may have no preference to M or G. Furthermore, Al-IV has primarily endolytic activity. See e.g., W. Hashimoto et al., Molecular Identification of Sphingomonas sp. Al Alginate Lyase (Al-IV) as a Member of Novel Polysaccharide Lyase Family 15 and Implications in Alginate Lyase Evolution, J. BIOSCIENCE AND BIOENGINEERING, Vol. 99, No. 1, 48-54, 2005; O. Miyake et al., An exotype alginate lyase in Sphingomonas sp. Al:

overexpression in Escherichia coli, purification, and characterization of alginate lyase IV (Al- IV), PROTEIN EXPRESSION & PURIFICATION, 29 (2003) 33-41; M. Yamasaki et al, Crystallization and preliminary X-ray analysis of alginate lyases Al-II and Al-ΙΓ from Sphingomonas sp. Al, ACTA CRYST. (2005). F61, 288-290; M. Yamasaki et al, A Structural Basis for Depolymerization of Alginate by Polysaccharide Lyase Family-7, J. MOL. BIOL. (2005) 352, 11-

[0056] In some embodiments of the methods described herein, the alginate lyase may be obtained from a recombinant source. In other embodiments of the methods described herein, the alginate lyase may include those listed in Table 1 below.

Table 1. Suitable lyases used for depolymerizing alginate

Protein 11) Source Mode of action

AF114040 Vibrio halioticoli Endo-lytic

AF114038 Vibrio halioticoli Endo-lytic

XP_002383604.1 Aspergillus flavus NRRL3357 Unknown

EDP53576.1 Aspergillus fumigatus A1163 Endo-lytic

EDP49916.1 Aspergillus fumigatus A1163 Endo-lytic

XP_748403.1 Aspergillus fumigatus Af293 Endo-lytic

CAK40226.1 Aspergillus niger Endo-lytic

BAE63841.1 Aspergillus oryzae Unknown

XP_566624.1 Cryptococcus neoformans var. neoform ans JEC21 Exo-lytic

XP_001729882.1 Malassezia globosa CBS 7966 Unknown

XP_002149780.1 Penicillium mameffei ATCC 18224 Unknown

XP_001552887.1 Botryotinia fucke liana B05.10 Unknown

XP_001839870.2 Coprinopsis cinerea okayama 7 #130 Unknown

XP_001878109.1 Laccaria bicolor S238N-H8 Unknown

EEU36914.1 Nectria haematococca mpVI 77-13-4 Unknown

EAW16796.1 Neosartorya fischeri NRRL 181 Unknown

XP_001597195.1 Sclerotinia sclerotiorum 1980 Unknown

XP_002340011.1 Talaromyces stipitatus ATCC 10500 Unknown

AB026618.1 Haliotis discus discus Exo-lytic

XP_778147.1 Cryptococcus neoformans var. neoform ans B-3501A Exo-lytic

BAD90006.1 Sphingomonas Endo-lytic

BAD16656.1 Sphingomonas Endo-lytic

BAB03319.1 Sphingomonas Exo-lytic

BAB03312.1 Sphingomonas Endo-lytic

ADE10038.1 Tremella fuciformis Exo-lytic

ZP_00991979.1 Vibrio splendidus Exo-lytic

CAA11481.1 Azotobacter chroococcum ATCC 4412 Endo-lytic

AAC04567.1 Azotobacter vinelandii Endo-lytic

AAC32313.1 Azotobacter vinelandii Endo-lytic

BAA33966.1 Cobetia marina N-l Endo-lytic

AAA71990.1 Pseudomonas aeruginosa 8830 Endo-lytic

AAA91127.1 Pseudomonas aeruginosa FRD1 Endo-lytic Protein 11) Source Mode of action

AAG06935.1 Pseudomonas aeruginosa PAOl Endo-lytic

NP_252237.1 Pseudomonas aeruginosa PAOl Endo-lytic

AAR23929.1 Pseudomonas sp. QD03 Endo-lytic

AAN63147.1 Pseudomonas sp. QDA Endo-lytic

AAF32371.1 Pseudomonas syringae pv. syringae FF5 Endo-lytic

2009330A Sphingomonas sp. AI Endo-lytic

BAA01182.1 Pseudomonas sp. OS-ALG-9 Endo-lytic

AC079344.1 Azotobacter vinelandii DJ ATCC BAA-1303 Endo-lytic

AC077598.1 Azotobacter vinelandii DJ ATCC BAA-1303 Endo-lytic

AC078583.1 Azotobacter vinelandii DJ ATCC BAA-1303 Endo-lytic

BAA83339.1 Corynebacterium sp. ALY-1 Endo-lytic

AAA25049.1 Klebsiella pneumoniae subsp. Aerogenes Unknown

CAA49630.1 Photobacterium sp. ATCC 43367 Unknown

AAG04556.1 Pseudomonas aeruginosa PAOl Unknown

NP_249858.1 Pseudomonas aeruginosa PAOl Unknown

BAD16656.1 Sphingomonas sp. AI Unknown

2009330A Sphingomonas sp. AI Unknown

AAF22512.1 Vibrio halioticoli IAM14596T Unknown

ABB36771.1 Vibrio sp. 02 Unknown

ABB36772.1 Vibrio sp. 02 Unknown

AAP45155.1 Vibrio sp. QY101 Unknown

BAE81787.1 Haliotis discus hannai Exo-lytic

BAC87758.1 Haliotis discus hannai Exo-lytic

BAB 19127.1 Chlorella virus CVK2 Unknown

BAA19848.1 Pseudomonas sp. OS-ALG-9 Unknown

AAD 16034.1 Pseudoalteromonas sp. IAM14594 Endo-lytic

AAK90358.1 Agrobacterium tumefaciens str. C58 Exo-lytic

BAI50574.1 Pseudoalteromonas atlantica AR06 Endo-lytic

1J1T[A] (PDB ID) Pseudoalteromonas sp. 272 Endo-lytic

AAD 16034.1 Pseudoalteromonas sp. IAM14594 Endo-lytic

ACB87607.1 Pseudoalteromonas sp. SM0524 Endo-lytic [0057] In certain embodiments, the alginate lyase is selected from XP_001839870.2,

XP_001878109.1, CAK40226.1, EAW16796.1, XP 748403.1, PDB: 2ZZJA, XP_566624.1,

BAE63841.1, XP_002383604.1, XP_002149780.1, EDP53576.1, EDP49916.1,

XP_002340011.1, XP_001552887.1, XP_001597195.1, XP_778147.1, XP_001729882.1,

EEU36914.1, AF082561c, AB018795, X70036,CAA58650.1, AF114039f, AF114037f,

AF114040f, AF114038f, XP_002383604.1, EDP53576.1, EDP49916.1, XP_748403.1,

CAK40226.1, BAE63841.1, XP_566624.1, XP_001729882.1, XP_002149780.1,

XP_001552887.1, XP_001839870.2, XP_001878109.1, EEU36914.1, EAW16796.1,

XP_001597195.1, XP_002340011.1, AB026618.1, XP_778147.1, BAD90006.1, BAD16656.1,

ADE10038.1, ZP_00991979.1, CAA11481.1, AAC04567.1, AAC32313.1, BAA33966.1,

AAA71990.1, AAA91127.1, AAG06935.1, NP_252237.1, AAR23929.1, AAN63147.1,

AAF32371.1, 2009330A, BAB03312.1, BAA01182.1, BAA83339.1, AAA25049.1,

CAA49630.1, AAG04556.1, NP_249858.1, BAD16656.1, 2009330A, BAB03312.1,

AAF22512.1, ABB36771.1, ABB36772.1, AAP45155.1, BAE81787.1, BAC87758.1,

BAB19127.1, BAA19848.1, AAD16034.1, and AAK90358.1.

[0058] It should be noted that one of skill in the art will understand that the lyase genes listed in Table 1 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each of the lyase genes listed in Table 1 above.

[0059] Additional suitable alginate lyases may be identified and isolated from organisms, such as fungi, that can grow as prototroph in media with alginate polymer as its sole carbon source. Methods for identifying and isolating proteins are well known in the art. In one example, cell extracts from a fungal species may be fractionated, for example by FPLC, to identify alginate lyases and other alginate metabolizing enzymes. The fractions can then be tested for alginate lyase activity by performing an alginate lyase activity assay. The protein responsible for lyase activity may then be identified by, for example, liquid

chromatography/tandem mass spectrometry (LC-MS/MS). Alternatively, cell-free extracts may be directly tested for alginate activity by measuring the rate of alginate degradation using a capillary viscometer. See e.g., Gimmestad et ah, The Pseudomonas fluorescens AlgG Protein, but Not Its Mannuronan C-5-Epimerase Activity, Is Needed for Alginate Polymer Formation, J. Bacteriol., Vol. 185, No. 12, pp. 3515-23, 2003. [0060] Another example for identifying and isolating a suitable alginate lyase includes performing a complementation or gain-of-function type screen. In another example, computationally mining of genomic DNA may be used to identify further alginate lyases. The sequencing may be done using, for example, the Illumina GS system. Assembly of the genome data may be done using any sequence analysis software known in the art. Additionally, suitable alginate lyases may be identified from alginate consuming organisms by mass spectrometry. In yet another example, microarray assays can be performed to identify candidates specifically expressed in alginate consuming organisms upon growth on media utilizing alginate as a sole carbon source to identify pathways and lyase genes for alginate metabolism.

Forming 4-deoxy-L-erythro-hexoseulose uronate (DEHU)

[0061] As depicted in reaction 100, once the alginate is depolymerized into oligoalginate, an oligoalginate lyase may be used to convert the alginate oligomers into monomers. Oligoalginate lyase 108 may be any exo-acting alginate lyases that can further depolymerize mixture 106 of oligosaccharides into monosaccharides.

[0062] Exo-acting alginate lyases typically act on the ultimate or penultimate glycosidic bond near the non-reducing end of the alginate polymer. Examples may include oligoalginate lyases (EC 4.2.2.-). The exo-acting alginate lyases catalyze further depolymerization of these oligosaccharides and release unsaturated monosaccharides, such as DEHU.

[0063] In some embodiments, the methods described herein employ an oligoalginate lyase isolated or partially purified from a recombinant source. The recombinant source may be from any eukaryote or prokaryote. In certain embodiments, the oligoalginate lyase may be obtained from bacteria, such as Escherichia coli, and Bacillus subtilis. The oligoalginate lyase may also be obtained from fungi, including for example, yeast. Such examples, may include,

Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, and Aspergillus sp.

Converting DEHU into 5-HPA

[0064] As depicted in reaction 100, DEHU can be spontaneously converted into 5-HPA in the presence of ammonia or an ammonium ion. In one embodiment, ammonia is added to the reaction mixture. In another embodiment, an ammonium ion is added to the reaction mixture. One skilled in the art would recognize that the ammonium ion may be added as an ammonium salt to the reaction to convert at least a portion of the DEHU into the 5-HPA. [0065] In some embodiments, the ammonium salt is ammonium chloride, ammonium phosphate, ammonium acetate, ammonium nitrate, ammonium sulfate, or a combination thereof.

In other embodiments, the ammonium salt is ammonium nitrate, ammonium sulfate, ammonium acetate, or ammonium chloride. In other embodiments, the ammonium salt is ammonium nitrate or ammonium sulfate. In yet other embodiments, the ammonium salt is ammonium phosphate, ammonium hydroxide or ammonium chloride. In one embodiment, the ammonium salt is ammonium chloride. In another embodiment, the ammonium salt is ammonium hydroxide.

[0066] In some embodiments, the ammonia or ammonium salt used in the reaction may have a concentration between 100 mM and 600 mM, or between lOOmM and 150 mM. In other embodiments, the ammonia or ammonium salt is added to the reaction mixture at a ratio of 0.32- 6.7 mole relative to 1 mole of DEHU.

[0067] In other embodiments, the conversion of DEHU into 5-HPA can be further accelerated in the presence of a phosphate ion. For example, DEHU can be converted to 5-HPA in the presence of ammonia or an ammonium ion and a phosphate ion at milder reaction conditions, including for example, at ambient temperature. The phosphate ion may be added in the form of a salt or an aqueous solution.

Isolating 5-HPA

[0068] One skilled in the art would recognize the various ways that 5-HPA could be isolated from the reaction mixture. For example, the product could be isolated by organic solvent extraction. Suitable solvents for the extraction include, for example, alkanes (e.g. , hexane), medium chain alcohols (e.g. , butanol and hexanol), and ethyl acetate. The product could also be isolated by column chromatography, for example, using silica gel.

[0069] In other embodiments, 5-HPA may be isolated as a solid by precipitation. The product may be precipitated by any suitable methods known in the art that can cause 5-HPA to form a solid (e.g. , crystal or pellet) from a solution. For example, 5-HPA may be precipitated by lowering the pH of the solution containing 5-HPA. In certain embodiments, 5-HPA may be isolated by precipitation at a pH of 4.5 or below.

[0070] The isolated 5-HPA may be further purified by recrystallization. In some

embodiments, 5-HPA may be recrystallized by lowering the pH of the solution containing 5- HPA. In certain embodiments, 5-HPA may be purified by recrystallization at a pH of 4.5 or below.

The Reaction Yield

[0071] Without wishing to be bound by theory, the methods described herein can result in higher- yielding reactions compared to the methods described in the art because the lyases make direct contact with the alginate outside of a cell to produce 5-HPA. Such a method can result in higher 5-HPA yields than a bacterium incorporating alginate into its cells to produce the compound, because the alginate is not consumed in alternate metabolic pathways that may compete with the production of 5-HPA.

[0072] In some embodiments, the methods described herein yield at least 32%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the theoretical maximum of 5-HPA that may be produced from the alginate used as the starting material. In yet other embodiments, the methods described herein yield between 32-99%, 50-99%, 55-99%, 60-99%, 75-99%, 80-99%, 50-95%, 55-95%, 60-95%, 75-95%, 80-95%, 50-90%, 55-90%, 60-90%, 75-90%, 80-90%, 50- 85%, 55-85%, 60-85%, 75-85%, 80-85%, 50-80%, 55-80%, 60-80%, 75-80%, 32%-65%, 40%- 65%, 45%-65%, or 50%-65% of the theoretical maximum of 5-HPA that may be produced from the alginate used as the starting material.

[0073] Furthermore, various reaction parameters have been optimized in the methods described herein to improve the reaction yield. Unless otherwise stated, "yield" as used herein refers to the theoretical maximum of 5-HPA that may be produced from the amount of starting materials used. For example, in some embodiments where alginate is the starting material, the yield refers to the theoretical maximum of 5-HPA that may be produced from the amount of alginate used. In other embodiments where oligoalginate is provided, the yield refers to the theoretical maximum of 5-HPA that may be produced from the oligoalginate provided. a) Reaction temperature

[0074] The methods described herein can, in some embodiments, produce 5-HPA from alginate at ambient conditions. It should be recognized, however, that the temperature of the reaction may depend on the enzymes used, and that the enzymes may become denatured at above certain temperatures. Further, the temperature of the reaction may also depend on the ions present in the reaction mixture.

[0075] As the reaction temperature increases, the percentage conversion into 5-HPA may increase. In some embodiments, the reaction temperature is between about room temperature (e.g., 18°C-20°C) and about 50°C. In some embodiments, the reaction temperature is between about 22°C and about 45°C. In other embodiments, the reaction temperature is between about 30°C and about 45°C. In yet other embodiments, the reaction temperature is between about 30°C and about 37°C. b) pH

[0076] The pH of the reaction may affect the stability of the alginate lyase and/or

oligoalginate lyase used in the methods described herein. It should be recognized, however, that the pH of the reaction may depend on the enzymes used. In some embodiments, the pH of the reaction is between about 6.0 to about 7.6. In other embodiments, the pH of the reaction is between about 7.2 to about 7.6. In one embodiment, the pH of the reaction is about 7.5. In yet other embodiments, the pH of the reaction is between about 4.5 to about 8.3. In yet other embodiments, the pH of the reaction is between about 6.5 to about 8.3.

[0077] In some embodiments, the pH of the formation of 5-HPA from DEHU is between about 4.5 and about 11. In other embodiments, the pH of the reaction to form 5-HPA from DEHU is between about 6.0 and about 9.0. In one embodiment, the pH of the reaction to form 5- HPA from DEHU is about 7.5.

Downstream Products

[0078] The 5-HPA produced according to the methods described herein can be converted into valuable industrial chemicals, including for example pyridine, piperidine and picolinic acid. For example, decarboxylation of 5-HPA can be thermally catalyzed to form 3-hydroxypyridine (3-HP). See e.g., Chemistry of Natural Compounds, Vol. 25, No. 1, pp. 125-126. The 3-HP can then be hydrogenolyzed to form piperidine. See e.g., J. Am. Chem. Soc, 1952, 74 (6), pp. 1485- 1488. Further, piperidine can be oxidized to form pyridine. See e.g., U.S. 2,765,311; New J. Chem., 2008, 32, p. 1027. EXAMPLES

[0079] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

Example 1: Conversion of sodium alginate into 5-HPA using an alginate lyase and a recombinant oligoalginate lyase in the presence of ammonium chloride

[0080] This example demonstrates the production of 5-HPA by degrading sodium alginate with a sigma alginate lyase and an oligoalginate lyase prepared from recombinant E. coli in M9 buffer.

Materials and Procedure

[0081] First, an oligoalginate lyase assay (1 mL) was prepared by combining the reagents listed below. This mixture was placed in a 37°C shaking incubator for about 48 hours.

• 150 of 10 x M9 buffer with pH 7.2

• Recipe for 1 x M9 buffer

o 64 mg/mL Na₂HP0₄-7H₂0

o 15 mg/mL KH₂P0₄

o 2.5 mg/mL NaCl

o 5 mg/mL NH₄C1

• 10 of 10 mg/ml sigma alginate lyase from Sigma Aldrich

• 2 f oligoalginate lyase crude lysate

• 1 50 mg/ml Kanamycin

• 1 50 mg/ml Chloramphenicol

• 1 100 mg/ml Ampicillin

• 2 alginate solution (pretreated with sigma alginate lyase)

• 61

7 Distilled water

[0082] The oligoalginate lyase crude lysate used in the oligoalginate lyase assay above was prepared as follows. E. coli BL21(DE3) strain harboring pETAtu_OAL was grown in Luria Bertani (LB) media overnight. Aliquot of this culture was inoculated into fresh LB media (10 mL) and was grown in an orbital shaker at 37 °C at 200 rpm. When the culture reached optical density at 600 nm of approximately 0.6, the culture was induced with isopropyl β-D-l- thiogalactopyranoside (IPTG), and further grown overnight in an orbital shaker at room temperature. The culture was then centrifuged. The resulting pellet was suspended in a

BugBuster cocktail (100 of protease inhibitor cocktail, 2 ase, 333

of pH 7.4, 0.15 mM of phosphate buffer, 150 of 2M sodium chloride, of water). The solution was chilled on ice for 30 minutes to lyse the cells. The cell lysate was centrifuged at 4300 rpm x g for 15 minutes, which resulted in the oligoalginate lyase solution.

[0083] Then, sodium alginate was converted into 5-HPA as follows. A 50 mL sample of fresh degraded alginate was prepared. 2.5 g of sodium alginate was weighed out and added to filter sterilized water. 5 mL of 10 x M9 stock (at pH 7.2) was added to the mixture, and then filtered. Sterilized water was added to a final volume of fifty mL in a 50 mL falcon tube. 50 μΐ_^ of 10 mg/mL sigma alginate lyase stock was added to the sodium alginate solution. This reaction proceeded for 24 hours. A crude oligoalginate lyase assay was then conducted. The assay was placed in a 30°C incubator for 24 hours. The first fraction of the sample was collected. The reaction was allowed to sit in the 30°C incubator for another 2 days before collecting a second fraction. The alginate did not appear to be fully degraded after the first 24- hour time point. As such, the reaction was allowed to incubate before the remaining fractions were collected. A sample having a concentration approximate between 12 -30 mM was obtained and analyzed.

Results

[0084] The formation of 5-HPA in the reaction mixture was confirmed by HPLC-UV, HPLC-MS, 1H NMR, and ¹³C NMR. By comparing the sample obtained from the reaction mixture to commercially available standards, it was confirmed that 5-HPA was produced. a) HPLC

[0085] Instrument parameters for HPLC-UV analysis:

• Column: 100 mM hypercarb column

• Mobile phase:

o solvent A: 0.2% trifluoroacetic acid (TFA) in HPLC grade water

o solvent B: HPLC grade methanol

• Mobile phase solvent gradient: 0 min - 30% solvent B, 15.00 min - 90% solvent B, 15.50 min -90% solvent B, 15.75-30% solvent B, and 21min-stop

• Flow rate 0.65 mL/min

• Run time: 21 min

• UV detector wavelengths: 210 nm, 235 nm

• Column oven: 65°C

• Injection volume: 5 μL

[0086] HPLC-UV: R_t = 7-8 min

[0087] Instrument parameters for HPLC-MS analysis: • Sim mode m/z: 140 and 172

• Event time: 0.1 sec

• Positive scanning

• DUIS interface

• DL temperature 250, Heat block temperature 400

[0088] HPLC-MS: R_t = 7-8 min b) NMR

[0089] A sample obtained from the reaction mixture was purified using a Shimadzu fraction collector before analysis by NMR.

[0090] Fraction-collection instrument parameters (HPLC):

• Column 100 mM hypercarb column

• Mobile phase:

o solvent A: 0.2% TFA in HPLC grade water

o solvent B: HPLC grade methanol

• Mobile phase solvent gradient: 0 min - 30% solvent B, 15.00 min - 90% solvent B, 15.50 min -90% solvent B, 15.75-30% solvent B, and 21min-stop

• Flow rate: 0.65mL/min

• Run time: 21 min

• UV detector wavelengths: 210 nm, 235 nm

• Column oven: 65C

• Injection volume: 50 μL

• Fraction Collection by Time: Valve open 5.50 min, Valve Closed 7.00 min.

• Fraction Collector Delay Volume: 650 μL

[0091] The sample was evaporated at 65°C using an evaporator. The sample was dissolved in DMSO-d₆ after evaporation, and placed in a 30°C shaking incubator to re-dissolve the sample in DMSO-d₆. The sample had a pH of 2 before evaporation. The sample was used for analysis by 1H NMR and ¹³C NMR.

[0092] 1H NMR (DMSO-d₆): δ 5.9 (s, OH), 7.3 (dd, 2H, CH₂), 7.95 (d, 2H, CH₂), 8.2 (d, 2H, CH₂). FIG. 2 depicts the peak assignments observed in this 1H NMR of the purified sample containing 5-HPA.

[0093] ¹³C NMR (DMSO-d₆): δ 123.24, 126.99, 137.83, 138.96, 157.27, 165.9.

[0094] Example 2: Conversion of sodium alginate into 5-HPA using alginate lyase and a recombinant oligoalginate lyase in the presence of ammonium sulfate [0095] This example demonstrates the production of 5-HPA from sodium alginate using an alginate lyase and a recombinant oligoalginate lyase in the presence of ammonium sulfate.

Materials and Procedure

[0096] A 2.5% sodium alginate solution was made from mixing 2.5 mL of 5% sodium alginate solution, 1.5 mL of filter sterilized water, 1 mL of 20 mL 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) buffer, and 5 μΐ_^ of 10 mg/mL sigma alginate lyase dissolved in M9 buffer. This sample was allowed to incubate for 24 hours in a 37°C shaking incubator.

[0097] 1 mL of this sample was used in an oligoalginate lyase assay containing:

• 3 mL of 20 mM HEPES buffer

• 200 μL of 10 mg/mL sigma alginate lyase in M9 buffer

• 400 μL of oligoalginate lyase crude lysate

• 20 μL of 50 mg/ml Kanamycin

• 20 μL of 50 mg/ml Chloramphenicol

• 20 μL of 100 mg/ml Ampicillin

• 4 mL above described alginate solution (pretreated with sigma alginate lyase)

• 12.34 mL filter sterilized water

[0098] This sample was allowed to incubate for 96 hours in a 30°C shaking incubator. After incubation 500 μΐ_^ of 50 g/L ammonium sulfate was added.

Results

[0099] The reaction mixture was analyzed by HPLC-MS using the instrument parameters described in Example 1 above. As seen in FIGS. 3 and 4, three peaks were observed: m/z 140 and m/z 172 correspond to 5-HPA; and m/z 175 corresponds to DEHU. A reduction of m/z 175 was observed along with an increase in 5-HPA, as depicted in FIG. 4. These results suggest that the addition of an ammonium ion significantly increased the conversion of DEHU into 5-HPA.

Example 3: Time course with ammonium ion addition

[0100] This example demonstrates the conversion of DEHU into 5-HPA in the presence of an ammonium ion, in which the DEHU is obtained from alginate. Materials and Procedure

[0101] The culture-free supernatant of a Bal 791 culture grown on 1% alginate and ammonium ion-free medium was added to 4% alginate to produce a DEHU-containing sample. To 5 mL of this sample, 500 μΐ_^ of 50 g/L ammonium sulfate was added. The sample was analyzed once right after ammonium sulfate had been added (i.e., the initial time point), and then again after about 5 hours (i.e., the end time point).

Results

[0102] As depicted in FIG. 5, the amount of 5-HPA produced in the sample increased over the 5 hours when ammonium sulfate was added to the reaction mixture. The results from this example also suggest that the spontaneous conversion of DEHU to 5-HPA can occur at room temperature (e.g., 18-22°C).

Example 4: Yield Determination

[0103] This example demonstrates a method to determine yield of 5-HPA produced. Materials and Procedure

[0104] First, a crude lysate of oligoalginate lyase producing E. coli strain was prepared as follows. With a sterile inoculating loop, 2

of glycerol stock from BAL stock #966 was transferred to 50 mL LB media containing 50ug/ml of kanamycin (Km50) in a 250 mL baffled shake flask. The culture was incubated at 37 °C with shaking (200 rpm) to an optical density measured at wavelength 600 nm (OD600) of 0.6 after about 4 hours. The expression of oligoalginate was induced by addition 50uL of 100 mM IPTG stock. The culture was then moved to a 25 °C incubator, and incubated with shaking at 200 rpm for 12 hours. 5 aliquots containing 10 mL of culture each was added to 15 mL centrifuge tubes. The aliquots were centrifuged at 4700 rpm for 20 minutes to pellet cells, and the supernatant was discarded. Each pellet from the 10 mL culture was re-suspended in about 1.12 mL BugBuster buffer (500uL 600mM NaCl, 500uL lOOmM PBS pH 7.4, lOOuL 10X BugBuster, 20 protease inhibitor cocktail). 2 of lysonase enzyme preparation was added, and the cells were allowed 30 minutes to lyse. The lysate was transferred to a microcentrifuge tube, and centrifuged at 13,000 rpm for 15 minutes. Cleared lysate (about lmL) was decanted into fresh a microfuge tube. [0105] Next, degraded alginate standards were prepared as follows. 250 mL of 1 x M9 buffer (at pH 5.5) was added to an Erlenmeyer flask. 30 g of sodium alginate (Sigma A2158) and 500 μΐ_^ of alginate lyase preparation were added to the flask. The contents in the flaks were mixed until all solids were dissolved. The contents were then diluted to 500 mL, and incubated at 37°C with shaking overnight. The alginate solution was centrifuged at 4700 rpm at 25°C for 4 hours. The centrifuged solution was then sterilized by vacuum filtration. Standards of various concentrations were created from the diluted 5% degraded sodium alginate with IX M9 buffer.

[0106] Then, an oligoalginate lyase buffer, and an oligoalginate lyase digest of the sample and standards were prepared as follows. Oligoalginate lyase degradation buffer master mix was prepared by assembling the reagents listed in Table 2 below in an Erlenmeyer flask with a stir bar (1 sample per 1 mL reaction). The reagents were sterilized by filtration through a 0.22 micron filter before addition to the flask.

Table 2. List of reagents used to prepare the oligoalginate lyase degradation buffer master mix

[0107] 950 μL· of the oligoalginate lyase buffer was then transferred to 1.5 mL centrifuge tubes. The liquid samples and standards were centrifuged. To these centrifuged samples and standards were added 200 μΐ_^ of sample/standard to 800 μΐ_^ oligoalginate lyase buffer, which were capped, vortexed and incubated at 37°C without shaking for 24 hours. The tubes were stored at 4°C until HPLC analysis.

[0108] To determine yield of the reaction, a comparison was made to standard curves of commercially available 5-HPA. Pure 5-HPA was obtained from DATO (minimum 98% pure). This product was a red crystal with an advertized molecular weight of 139.11. A carboxylate sodium salt of 5-HPA was obtained from Indofine Chemical Company (minimum 98% pure). This product was a light green powder. Standard solutions (approximately 0.2%) of 5-HPA from both vendors were prepared using a lOmL volumetric flask. Standard dilutions were prepared for a sample obtained from the reaction mixture, and the two commercially available 5-HPA samples. Each sample was injected on an HPLC column (10 μΐ_^ injection, detection at 210 nm) to obtain the graph depicted in FIG. 6.

Analysis

[0109] The DATO standard was considered as a standard for comparison to determine yield of 5-HPA produced in the reaction. From this experiment, the percent conversion from alginate to 5-HPA was calculated to be 74.0 % of the maximum theoretical yield.

Example 5: Optimization of Reaction Temperature

[0110] This example demonstrates the effect of different reaction temperatures on the yield of 5-HPA produced from sodium alginate.

Materials and Procedure

[0111] Based on the materials and procedures described in Example 4 above, four separate reactions with a sample size of 2.5 mL (total volume) were set up in 15 mL Falcon tubes. The sodium alginate concentration for each reaction was 0.15% (i.e., 5: 1 dilution of 0.75% degraded alginate). The four reactions were at different temperatures: room temperature (i.e., about 22°C), 30°C, 37°C and 45°C.

Analysis

[0112] As depicted in FIG. 7, the reactions reached approximately the same endpoint between 30-45°C. The reaction was completed within 18 hours at a temperature between 30- 45°C. Thus, the reaction to convert sodium alginate into 5-HPA may be performed between room temperature and 45°C, and preferably between 30-45°C.

Example 6: Optimization of Reaction pH

[0113] This example demonstrates the effect of different reaction pH on the yield of 5-HPA produced from sodium alginate.

Materials and Procedure

[0114] Based on the materials and procedures described in Example 4 above, eleven separate reactions with a sample size of 2.5 mL (total volume) were set up. The eleven reactions were performed at different pH values: 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.0 with the addition of 20 mM ammonium chloride.

[0115] The sodium alginate concentration for each reaction was 0.2%. The reactions took place at 37°C without shaking. The reactions were buffered with IX M9, and titrated to achieve various pH values between 6.0 and 7.6. The M9 buffer contained 19 mM of ammonium chloride.

Analysis

[0116] As depicted in FIG. 8, the alginate lyase and oligoalginate lyase digest was observed to be effective between a pH ranging from 6.0 to 7.6. The fastest initial rate was observed at pH 7.4. The highest signal at endpoint was observed for the reaction at pH 6.8, although all signals at the endpoint were within +10%. Thus, the reaction to convert sodium alginate into 5-HPA may be performed at pH 6.0-7.6.

Example 7: Optimization of Phosphate Concentration, Alginate Concentration, and pH

[0117] This example demonstrates the effect of different phosphate and alginate

concentrations, and different pH on the conversion of alginate into 5-HPA.

Materials and Procedure

[0118] Both 0.4 weight% by volume and 1.0 weight% by volume sodium alginate solutions were degraded with Sigma alginate lyase and oligoalginate lyase in the presence of ammonia to produce 5-hydroxypyridine-2-carboxylic acid. This degradation and production was tested in 100 mM phosphate, 50 mM phosphate and 10 mM phosphate solutions at pH 4.5 to 8.3 in about 0.5 pH unit increments. Table 3 below summarizes the reaction conditions in this example.

Table 3. Conditions for reaction series A to F

Analysis

[0119] Table 4 below summaries the pH at the beginning and at the end of each reaction in series A-F. Precipitation of 5-HPA was observed at pH values below 4.5, which may account for lower 5-HPA yields in the supernatant observed at lower pH values.

Table 4. pH of each sample tested

[0120] As shown in FIG. 9, at a pH below 6.5, enzymatic activity was observed to decrease. At a pH 7.0 7.7 with 100 mM phosphate and 0.4% alginate, the reaction was observed to yield the highest amount of 5-HPA (91% yield).

Example 8: Optimization of Phosphate Concentration, DEHU Concentration, and pH in the conversion of pre-degraded alginate into 5-HPA

[0121] This example demonstrates the effect of different phosphate and alginate

concentrations, and different pH on the conversion of pre-degraded alginate into 5-HPA.

Materials and Procedure

[0122] To alginate that had been degraded at pH 7.0 in 5 mM 3-(N- morpholino)propanesulfonic acid (mops) buffer, ammonium chloride was added with varying phosphate concentrations, alginate concentrations and pH. The reactions were performed in 100 mM phosphate, 50 mM phosphate and 10 mM phosphate solutions at pH 4.5 to 8.0 in 0.5 pH- unit increments. Table 5 below summarizes the reaction conditions in this example.

Table 5. Conditions for reaction series A to F Series Phosphate Alginate Ammonium chloride concentration (mM) concentration concentration (mM)

(weight %/volume)

A series 100 0.4 188

B series 50 0.4 188

C series 10 0.4 188

D series 100 1.0 188

E series 50 1.0 188

F series 10 1.0 188

Analysis

[0123] Table 6 below summaries the pH at the beginning and at the end of each reaction in series A-F. Precipitation of 5-HPA was observed at pH values below 4.5, which may account for lower yields observed at these pH values.

Table 6. pH of each sample tested

[0124] As shown in FIG. 10A, an 82% yield was observed at pH 6.5 with 100 mM phosphate and 0.4 weight % by volume alginate. At pH 6.0, 7.0 and pH 7.5 under these same conditions, about an 80% yield was observed. It was observed that phosphate catalyzed the formation of 5- HPA from DEHU around a neutral pH. Thus, a higher phosphate concentration, as well as a lower alginate concentration, were observed to produce higher yields of 5-HPA.

Example 9: Optimization of Phosphate Concentration, DEHU Concentration, and pH in the conversion of pre-degraded alginate into 5-HPA [0125] This example demonstrates the effect of different phosphate and alginate concentrations, and different pH on the conversion of pre-degraded alginate into 5-HPA.

Materials and Procedure

[0126] To alginate that had been degraded at pH 7.0 in 5 mM 3-(N- morpholino)propanesulfonic acid (mops) buffer, ammonium chloride was added with varying phosphate concentrations, alginate concentrations and pH. The reactions were performed in 100 mM phosphate and 10 mM phosphate solutions at pH 8.0 to 11.0 in 1.0 pH-unit increments. Table 7 below summarizes the reaction conditions in this example.

Table 7. Conditions for reaction series A, B, E and F

Analysis

[0127] Table 8 below summaries the pH at the beginning and at the end of each reaction in series A, B, E and F.

Table 8. pH of each sample tested

[0128] As shown in FIG. 10B, a 58% yield was observed at pH 8.0 with 100 mM phosphate and 0.4 weight % by volume alginate. At pH 9.0 under these same conditions, about a 55% yield was observed. It was observed that phosphate catalyzed the formation of 5-HPA from DEHU around a neutral pH. Thus, a higher phosphate concentration, as well as a lower alginate concentration, were observed to produce higher yields of 5-HPA.

Example 10: Optimization of Ammonium Chloride (NH₄CI) Concentration in the

Oligoalginate Lyase Digest

[0129] This example demonstrates the effect of different ammonium chloride concentrations on the yield of 5-HPA produced from sodium alginate.

Materials and Procedure

[0130] Based on the materials and procedures described in Example 4 above, two separate reactions with a sample size of 2.5 mL (total volume) were set up. The oligoalginate lyase digest was performed with various initial concentrations of NH₄CI. The ammonium concentration in IX M9 was about 9.5 mM. Between 0-30 mM NH₄CI was added to the reaction mixtures, and 4.5 mM of DEHU was present in the reaction mixtures of this experiment. In other words, between 0-6.7 mole of NH₄C1 was added relative to 1 mole of DEHU. The 5-HPA signal was tracked over time.

Analysis

[0131] As seen in FIG. 11A, a 44% increase in reaction rate (delta signal/time) was observed with the addition of 20 mM NH₄C1 when the reaction was performed at pH 7. As seen in FIG. 11B, addition of between 10-30mM {i.e., 2-6.7 equiv of NH₄C1 to 1 mole of DEHU) was not observed to significantly change the yield of 5-HPA.

Example 11: Conversion of sodium alginate from various sources into 5-HPA using an alginate lyase and an oligoalginate lyase

[0132] This example demonstrates the production of 5-HPA obtained from various sources using an alginate lyase and an oligoalginate lyase.

Materials and Procedure

[0133] 50mL solution of approximately 2% sodium alginate/lX M9 pH 5.5 was prepared using various alginates commercially available from Sigma Aldrich. Polymeric alginate was degraded overnight at 37 °C using Sigma alginate lyase (10 μg/mL). Three additional standard dilutions were prepared for each alginate solution. These solutions were subjected to an oligoalginate lyase, and the reaction mixture was analyzed via HPLC.

Analysis

[0134] As shown in FIG. 12, all alginate standards gave nearly identical 5-HPA signals, despite slightly dissimilar coloration and texture. DATO 5-HPA standards were also re-run with this batch. The percent conversion was calculated to be 72.9% on average for all alginate standards.

Example 12: Conversion of sodium alginate into 5-HPA using various alginate lyases and oligoalginate lyases

[0135] This example demonstrates the production of 5-HPA using different alginate lyases and oligoalginate lyases.

Materials and Procedure

[0136] 50mL solution of approximately 2% sodium alginate/lX M9 pH 5.5 was prepared using alginate commercially available from Sigma Aldrich. Polymeric alginate was degraded overnight at 37 °C using Sigma alginate lyase (10 μg/mL) or recombinant alginate lyases over- expressed and prepared from E. coli C strain harboring pCC-Ag43-SM0524 (SM0524) and E. coli BL21(DE3) strain harboring pETAl-1 (Al-1). The gene encoding alginate lyase SM0524 and Al-1 were derived from Pseudoalteromonas sp. SM0524 and Sphingomonas sp. Al (Al-1), respectively. These solutions were subjected to an oligoalginate lyase from BAL #966, and the reaction mixture was analyzed via HPLC.

[0137] Three additional standard dilutions were prepared from degraded alginate solution using Sigma alginate lyase. These solutions were subjected to different oligoalginate lyases, and the reaction mixture was analyzed via HPLC. The recombinant oligoalginate lyases were over- expressed and isolated from BAL stock #966 and E. coli BL21 harboring a pETV12B01_24239 vector (OAL derived from Vibrio splendidus 12B01).

Analysis

[0138] As shown in FIG. 13, different alginate lyases SM0524 and Al-1 can also generate 5- HPA and 10% better yield than Sigma alginate lyase. Additionally, as shown in FIG. 14, although the yield was approximately half, oligoalginate lyase derived from Vibrio splendidus

SM0524 was also observed to generate 5-HPA.

Example 13: Optimizing the ammonium chloride (NH₄CI) concentration and pH in the conversion of sodium alginate into 5-HPA

[0139] This example demonstrates the effect of different ammonium chloride concentrations and different pH on the yield of 5-HPA produced from sodium alginate.

Materials and Procedure

[0140] All reactions were carried out at 30°C. Stirring was set to 100-250 rpm, depending on the viscosity of the solution. The pH was set to 7.5 and controlled by addition of 5N ammonium hydroxide (NH₄OH). The final solid loading of sodium alginate was about 80-90 g/L.

[0141] First, 1800mL of 110 g/L sodium alginate (plus 0.005% sodium azide) was degraded at 37°C for 48 hours with 20 mg/mL Sigma alginate lyase. After 48 hours, 21.4 g sodium phosphate (Na₂HP0₄) (lOOmM) was added to the reaction mixture, and the pH of the solution was adjusted to 6.9 with 30 mL NH₄OH. The degraded alginate was divided among three reactors.

[0142] In the first reactor, 178 g (550mM) NH₄C1 was added, along with 0.6 mL of 10 mg/mL Sigma alginate lyase and 2 mL oligoalginate lyase cleared lysate (from LB culture pellet). The pH of the solution was adjusted to 6.5 with approximately 5 mL of 5N phosphoric acid.

[0143] In the second reactor, 36 g (1 lOmM) NH₄C1 was added, along with 0.6 mL of 10 mg/mL Sigma alginate lyase and 2 mL oligoalginate lyase cleared lysate (from LB culture pellet). The pH of the solution was adjusted to 7.5 using approximately 5 mL of 5N NH₄OH.

[0144] In the third reactor, 178 g (550mM) NH₄C1 was added, along with 0.6 mL of 10 mg/mL Sigma alginate lyase and 2 mL oligoalginate lyase cleared lysate (from LB culture pellet). The pH of the solution was adjusted to 7.5 using approximately 5 mL of 5N NH₄OH.

[0145] At 13 hours, 2mL of oligoalginate lyase cleared lysate (from LB culture pellet) was added to each reactor. At 21 hours, 5 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reactor. At 43 hours, 5 mL of oligoalginate lyase cleared lysate (from

TB culture pellet) and 0.5 mL 10 mg/mL Sigma alginate lyase were added to each reactor.

Analysis

[0146] In this example, the initial concentration of NH₄C1 and pH was varied. A titer of 51.5 g/L of 5-HPA (79.8% yield) was achieved in 150 hours using a 110 mM concentration of NH₄C1. A titer of 43.8 g/L of 5-HPA (73.8% yield) was achieved in 180 hours using the higher initial concentration of NH₄C1. As shown in FIGS. 15A and 15B, it was observed that increasing the concentration of NH₄C1 from 110 mM to 550 mM decreased the initial reaction rate by approximately 2-fold. It was also observed that decreasing the pH from 7.5 to 6.5 decreased the initial reaction rate by approximately two-fold. Thus, lower ammonium salt concentrations and a higher pH (about 7.5) may cause a faster conversion of sodium alginate into 5-HPA.

Example 14: Optimizing the temperature in the conversion of sodium alginate into 5-HPA

[0147] This example demonstrates the effect of different reaction temperatures on the yield of 5-HPA produced from sodium alginate.

Materials and Procedure

[0148] 1200 mL of 225 g/L sodium alginate (plus 0.005% sodium azide) was prepared by degrading a 12.5% sodium alginate solution using 1.2 mL 10 mg/mL Sigma alginate lyase for 24 hours. The final solid loading of sodium alginate was about 175 g/L.

[0149] After 24 hours, 75 grams of sodium alginate was added, along with 2 mL 10 mg/mL Sigma alginate lyase. After 48 hours, 45 grams sodium alginate was added. After 72 hours, 13.8 grams of ammonium phosphate (NH₄H₂P0₄) (100 mM) was added, along with 3 mL 10 mg/mL Sigma alginate.

[0150] The pH of the solution was adjusted to 7.5 with approximately 30 mL 5N NH₄OH. 25 mL oligoalginate lyase cleared lysate (from 5 mL TB culture pellet) was added to bring the total volume of the solution to 1200 mL. This solution was split into two bioreactors: the first for an ambient reaction, and the second for a 30°C temperature reaction. The pH was set to 7.5 and controlled by addition of 5N of NH₄OH.

[0151] At 71 hours, 10 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reactor. At 122 hours, 12.5 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reactor. At 231 hours, 15 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reactor.

Analysis

[0152] In this example, the reaction temperature was varied from ambient (about 18-22°C) to 30°C. A titer of 80.6 g/L 5-HPA and a yield corresponding to 65.7% of the theoretical maximum was achieved in 408 hours for the reaction at 30C. As shown in FIG. 16, the reaction at ambient temperature proceeded more slowly (i.e., about two-fold more slowly), achieving a titer of 75.4 g/L of 5-HPA (61.4% yield). Thus, increasing the reaction temperature may result in higher titers, yields, and reaction rates.

Example 15: Production of 5-HPA from Macroalgae

[0153] This example demonstrates the production of 5-HPA from macroalgae (i.e., seaweed).

Materials and Procedure

[0154] To a first reactor, 100 grams of Macrocystis pyrifera (also known as M. pyrifera) (250 g/L) was degraded at 37°C for 24 hours in approximately 660 mL of de-ionized water containing 5 mL 5% sodium azide, 1 mL CTec2 (a cocktail mix of enzymes from Novozymes), 0.2 mL HTec (a cocktail mix of enzymes from Novozymes) and 1 mL 10 mg/mL Sigma alginate lyase.

[0155] To a second reactor, 100 grams of Saccharina japonica (also known as S. japonica) (200 g/L) was degraded at 37 °C for 24 hours in approximately 660 mL of de-ionized water containing 5 mL 5% sodium azide, 1 mL CTec2, 0.2 mL HTec and 1 mL 10 mg/mL Sigma alginate lyase. After 24 hours, 50 grams of S. japonica was added to the second reactor.

[0156] After 48 hours, 50 grams of macroalgae was added to each reactor. After 96 hours, the solutions in each reactor were centrifuged to 4 hours at 4700 rpm. Supernatants were decanted and saved in separate media bottles. About 300 mL of 10 ug/mL Sigma alginate lyase was used to re-suspend each pellet, and these solutions were incubated for 24 hours at 37°C. After 24 hours, these solutions were centrifuged for 4 hours and 4700, rpm and combined with the first set of supernatants. [0157] 6.9 grams (100 mM) of ammonium phosphate (NH₄H₂P0₄) was added to 600mL of each degraded macroalgae supernatant, along with 10 mL of oligoalginate lyase cleared lysate from terrific broth (TB) culture pellet. The reaction temperature for each reaction was set to

30°C, and the pH was set to 7.5 and controlled by addition of 5N ammonium hydroxide

(NH₄OH).

[0158] At 71 hours, 2.5 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reaction. At 122 hours, 2.5 mL of oligoalginate lyase cleared lysate (from TB culture pellet) was added to each reaction.

Analysis

[0159] In this example, 5-HPA production was achieved directly from degraded macroalgae. As shown in FIG. 17, 17.7 g/L of 5-HPA was produced from S. japonica (68.7% yield), and 8.0 g/L 5-HPA was produced from M. pyrifera (54.2% yield). Thus, 5-HPA can be produced directly from macroalgae.

Example 16: Production of 5-HPA Using a Fed-Batch Process

[0160] This example demonstrates the production of 5-HPA from sodium alginate using a fed-batch process.

Materials and Procedure

[0161] 600mL of 100 mM ammonium phosphate (NH₄H₂P0₄) containing 10 ug/mL of Sigma alginate lyase and lOmL of oligoalginate lyase cleared lysate (from TB culture pellet) was prepared. To this solution, 50 grams sodium alginate was added. The reaction temperature was set to 30°C and the pH was set to 7.5, controlled by addition of 5N ammonium hydroxide (NH₄OH). The reaction was performed using a fed-batch process.

[0162] At 15 hours, it was observed that the pH had dropped to 5.8 due to the base pump. As such, the base pump was adjusted and 25 grams of sodium alginate was added to the reaction.

[0163] At 108 hours, 10 mL oligoalginate lyase cleared lysate was added to the reaction. At 117 hours, 20 grams sodium alginate was added to the reaction. At 204 hours, 10 grams sodium alginate was added to the reaction. Analysis

[0164] In this example, 5-HPA was produced directly from polymeric sodium alginate in a fed-batch process. This process achieved a high titer of 5-HPA with relatively low enzyme loading. As shown in FIG. 18, 67.6 g/L of 5-HPA was produced (73.5% yield).

Example 17: Purification of 5-HPA

[0165] This example demonstrates the purification of 5-HPA under acidic conditions, at a pH of 4.5. Since 5-HPA has been observed to have decreased solubility at pH values below 4.5, it was hypothesized that 5-HPA production at acidic pH values may be useful for instant purification.

Materials and Procedure

[0166] To alginate pre-degraded at pH 7.0 in 5 mM 3-(N-morpholino)propanesulfonic acid (mops) buffer, ammonium chloride was added with varying phosphate concentrations, alginate concentrations and ammonium chloride concentrations.

[0167] Table 9 below summarizes the reaction conditions in this example. Solubility was about 2 g/L under these reaction conditions.

Table 9. Reaction conditions for series G to J

Analysis

[0168] As seen in FIG. 19A, an ammonium chloride concentration of 656 mM produced an overall higher yield of 5-HPA in the supernatant than an ammonium chloride concentration of 188 mM. However, a precipitated solid (i.e., a pellet) was observed in some of the reactions.

When the pellet was re-dissolved into the supernatant, no significant difference in 5-HPA yield was observed as seen in FIG. 19B. Thus, a higher ammonium concentration increased the solubility of 5-HPA.

Example 18: 5-HPA Solubility Study

[0169] This example illustrates the solubility of 5-HPA to simply the purification process of this compound.

Materials and Procedure

[0170] Samples of 5 weight % by volume and 1 weight by volume of 5-HPA from Dato Chemical Company were prepared by dissolving 5-HPA in basic solution. To these solutions 2M hydrochloric acid was added in varying amounts in order to precipitate out the 5-HPA. The final pH was measured by using a pH strip.

[0171] Table 10 below compares the expected and observed concentrations of 5-HPA with yield and pH values observed.

Table 10. Comparison of expected versus observed concentration of 5-HPA with yield and pH

Analysis

[0172] From a pH range of 1 and 4.5, solubility was observed to decrease between 3 g/L and 1 g/L in both the 5 weight % by volume and 1 weight % by volume solutions. Thus, decreasing the pH of a 5-HPA solution can cause precipitation of 5-HPA as a solid.

Example 19: Production of 5-HPA from Undaria pinnatifida

[0173] This example demonstrates the production of 5-HPA from Undaria pinnatifida, which is a form of macroalgae (i.e., seaweed).

Materials and Procedure

[0174] 100 grams of Undaria pinnatifida (also known as wakame) (100 g/L) was degraded at 30°C for 44 hours in de-ionized water containing 5 mL 5% sodium azide, 2 mL CTec2 (a cocktail mix of enzymes from Novozymes), 0.5 mL HTec (a cocktail mix of enzymes from Novozymes) and 1 mL 60 mg/L Sigma alginate lyase, 1.120 mg/L of oligoalginate lyase cleared lysate from terrific broth (TB) culture pellet of BAL stock #966, and 20 g/L dibasic ammonium phosphate. The pH was set to 7.5 and controlled by addition of 5N ammonium hydroxide (NH₄OH).

Analysis

[0175] In this example, 5-HPA was observed to be produced directly from Undaria pinnatifida. 12.9 g/L of 5-HPA was observed to be produced from U. pinnatifida (45% yield).

Example 20: Production of 5-HPA from Saccharina latissima and Ascophylum nodosum

[0176] This example demonstrates the production of 5-HPA from Saccharina latissima and Ascophylum nodosum, which are forms of brown algae.

Materials and Procedure

[0177] To a first reactor, 75 mg of Saccharina latissima (7.5 g/L) was degraded at 37°C for 24 hours in approximately 365 μΐ_^ of de-ionized water containing 5 μΐ_^ 5% sodium azide, 100 μΐ_^ of 10 x M9 stock (at pH 7.2), and 1 mL 10 mg/mL Sigma alginate lyase. The 10 x M9 stock (at pH 7.2) included: dibasic sodium phosphate (60.0g/L), monobasic potassium phosphate

(30.0g/L), sodium chloride (5.0 g/L) and ammonium chloride (10.0 g/L). [0178] To a second reactor, 75 mg of Ascophylum nodosum (7.5 g/L) was combined with 5 mM ethylene diamine tetra-acetate (EDTA) for 1 hour prior to digestion using alginate lyase. After incubation with the EDTA, approximately 365

of de-ionized water containing 5 5% sodium azide, 100 of 10 x M9 stock (at pH 7.2), and 1 mL 10 mg/mL Sigma alginate lyase were added to the second reactor to degrade the Ascophylum nodosum at 37 °C for 24 hours.

[0179] After 24 hours, the reaction solutions in each reactor were centrifuged to obtain a degraded macroalgae supernatant. 200 of 10% NH₄C1 was added to 10 mL of each degraded macroalgae supernatant, along with 20

of cleared lysate from TB cultured BAL #966 pellet. The reaction temperature was set to 30°C, and the pH was set to 7.5.

Analysis

[0180] 5-HPA was observed to be produced directly from Saccharina latissima at a yield of 0.0986 g of 5-HPA per g of dry seaweed. 5-HPA was observed to be produced directly from Ascophylum nodosum at a yield of 0.2053 g of 5-HPA per g of dry seaweed.

Claims

1. A method of producing 5-hydroxypyridine-2-carboxylic acid (5- HPA), the method comprising: a) providing oligoalginate; b) providing an isolated oligoalginate lyase; c) contacting the oligoalginate with the isolated oligoalginate lyase to form 4- deoxy-L-erythro-5-hexoseulose uronate (DEHU); and d) converting at least a portion of the DEHU to 5-HPA.

2. The method of claim 1, further comprising isolating the 5-HPA.

3. The method of claim 1 or 2, wherein the oligoalginate is obtained

from Saccharina japonica, Macrocystis pyrifera, Saccharina

latissima, Saccharina digitata, Undaria pinnatifida, Ascophyllum

nodosum, Saccharina angustata, or Sargassum muticum.

4. The method of any one of claims 1-3, wherein the oligoalginate

lyase is obtained from a recombinant source.

5. The method of any one of claims 1-4, wherein the oligoalginate

lyase is obtained from E. coli or yeast.

6. The method of any one of claims 1-5, further comprising adding

ammonia or an ammonium salt, wherein the addition of ammonia

or an ammonium salt converts at least a portion of the DEHU into

the 5-HPA.

7. The method of claim 6, wherein the ammonium salt is selected

from the group consisting of ammonium chloride, ammonium

phosphate, ammonium acetate, ammonium nitrate, ammonium

sulfate, and ammonium hydroxide, or a combination thereof.

8. The method of claim 7, wherein the ammonium salt is ammonium

chloride or ammonium hydroxide.

9. The method of claim 6, wherein the ammonia or ammonium salt is

added at a ratio of 0.32-6.7 mole relative to 1 mole of DEHU.

10. The method of claim 6, further comprising adding a phosphate salt

to convert at least a portion of the DEHU into the 5-HPA.

11. The method of any one of claims 2-10, wherein the isolating of the

5-HPA comprises lowering the pH to 4.5 or below to precipitate

the 5-HPA.

12. The method of claim 11, further comprising purifying the isolated

5-HPA by precipitation.

13. The method of claim 12, wherein the precipitation is performed at

a pH of 4.5 or below.

14. The method of any one of claims 1-13, further comprising

converting the 5-HPA into one or more compounds selected from

the group consisting of pyridine, piperidine and picolinic acid.

15. A method of producing 5-hydroxypyridine-2-carboxylic acid (5- HPA), the method comprising: a) providing alginate; b) providing an isolated alginate lyase; c) providing an isolated oligoalginate lyase; d) contacting the alginate with the isolated alginate lyase to form oligoalginate; e) contacting the oligoalginate with the isolated oligoalginate lyase to form 4- deoxy-L-erythro-5-hexoseulose uronate (DEHU); and f) converting at least a portion of the DEHU to 5-HPA.

16. The method of claim 15, further comprising isolating the 5-HPA.

17. The method of claim 15 or 16, wherein the alginate has a

concentration of about 1-100 g/L.

18. The method of claim 17, wherein the alginate has a concentration of about 5-50 g/L.

19. The method of any one of claims 15-18, wherein the alginate is sodium alginate.

20. The method of claim 19, wherein the alginate is a polymeric

sodium alginate.

21. The method of any one of claims 15-20, wherein the alginate is obtained from Saccharina japonica, Macrocystis pyrifera, Saccharina latissima, Saccharina digitata, Undaria pinnatifida, Ascophyllum nodosum, Saccharina angustata, or Sargassum muticum.

22. The method of any one of claims 15-21, wherein the alginate lyase is obtained from a recombinant source.

23. The method of any one of claims 15-22, wherein the oligoalginate lyase is obtained from a recombinant source.

24. The method of any one of claims 15-23, wherein the oligoalginate lyase is obtained from E. coli or yeast.

25. The method of any one of claims 15-24, further comprising adding ammonia or an ammonium salt, wherein the addition converts at least a portion of the DEHU into the 5-HPA.

26. The method of claim 25, wherein the ammonium salt is selected from the group consisting of ammonium chloride, ammonium phosphate, ammonium acetate, ammonium nitrate, ammonium sulfate, and ammonium hydroxide, or a combination thereof.

27. The method of claim 26, wherein the ammonium salt is ammonium chloride or ammonium hydroxide.

28. The method of any one of claims 25-2 ^', wherein the ammonia or ammonium salt is added at a ratio of 0.32-6.7 mole relative to 1 mole of DEHU.

29. The method of claim 25, further comprising adding a phosphate salt to convert at least a portion of the DEHU into the 5-HPA.

30. The method of any one of claims 15-28, wherein the method yields at least 32% of the theoretical maximum of 5-HPA that may be produced from the alginate.

31. The method of claim 30, wherein the method yields between 50% to 80% of the theoretical maximum of 5-HPA that may be produced from the alginate.

32. The method of any one of claims 16-31, wherein the isolating of the 5-HPA comprises lowering the pH to 4.5 or below to precipitate the 5-HPA.

33. The method of any one of claims 16-32, further comprising

purifying the isolated 5-HPA by precipitation.

34. The method of claim 33, wherein the precipitation is performed at a pH of 4.5 or below.

35. The method of any one of claims 15-34, further comprising

converting the 5-HPA into one or more compounds selected from the group consisting of pyridine, piperidine and picolinic acid.