Conversion of lysine phosphate to 1.5 pentanediamine and separation of the diamine from amino acids and carboxylic acids

Abstract

A method for producing 1,5 pentanediamine (also known as PDA, pentamethyldiamine, PMDA and cadaverine) by decarboxylation of lysine using lysine decarboxylase in the presence of sulfate or more preferably phosphate anion salts of lysine is described. The process works at high pH where decarboxylases do not function, using HCL salts of lysine, and at substrate concentrations that cannot be achieved with lysine HCL at the usual pH optimum of carboxylases. Furthermore, a chromatographic process for purifying PDA from lysine and conjugated anions of lysine salts is described, which can be applied as a general process for purifying any diamine from a product mixture containing amino acids and / or carboxylic acids with their conjugated anions, and which uses a strong anion exchange resin as the stationary phase. The method does not utilize any organic solvent and has been shown to be economical and efficient for industrial-scale isolation of diamines from complex mixtures containing amino acids and / or carboxylic acids and conjugated anions from amine salts.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to a process for separating diamines from a mixture comprising a diamine and one or more amino acids and / or carboxylic acids at low cost and in high yield without the use of any organic solvent. [Background technology]

[0002] background Diamines are used in the synthesis of polyamides and polyurethanes. For example, 1,4-diaminobutane and 1,6-diaminohexane are used in the synthesis of nylon-4,6 and -6,6, respectively. Diamines can be produced by biochemical or chemical methods. Biochemical methods employ cultures of transformed microorganisms that have been engineered to secrete diamines into the medium or amino acid decarboxylases into medium containing amino acids (see, e.g., U.S. Pat. Nos. 11,124,812, 11,053,524, 11,053,525, 10,626,425, 10,870,871, 10,711,289, 10,640,798, 10,472,636, 10,150,977, 9,745,608, 9,644,220, 9,365,876, 9,115,362, and 8,741,623, each of which is incorporated herein by reference in its entirety). In the case of microorganisms that secrete amino acid decarboxylases (see, for example, U.S. Patent No. 8,871,477, which is incorporated herein by reference in its entirety), amino acids such as lysine or ornithine are added to the clarified medium of the biomass to produce diamines by the action of the decarboxylases. In some methods, diammonium salts of carboxylic acids are used to produce diamines (see, for example, U.S. Patent No. 8,742,060, which is incorporated herein by reference in its entirety). In any of the above methods, the crude product is a complex mixture containing amino acids and / or carboxylic acids from the amino acids and / or carboxylic acids used to produce diamines, and the medium usually contains one or more carboxylic acid metabolites, such as acetate, oxalate, succinate, tartarate, lactic acid and / or salts thereof, as well as conjugated ions of amine or carboxylic acid functional groups, such as phosphate, sulfate, hydrochloride, chloride, ammonium, sodium, potassium, etc., which may be present due to the addition of mineral nutrients in the medium. Therefore, efficient separation of diamines from such complex mixtures in high yield and purity and at low cost continues to be a challenging process.

[0003] Several methods have been reported for isolating and purifying diamines such as 1,5-pentanediamine from reaction mixtures. Japanese Patent No. 581215 and Japanese Patent Publication No. 2016033138, each of which is incorporated herein by reference in its entirety, disclose a distillation method for isolating 1,5-pentanediamine from a mixture obtained from a reaction mixture of pyrolysis or enzymatic conversion. Several methods have been reported to isolate 1,5-pentanediamine from a reaction mixture by adsorbing the diamine onto a neutral or anionic resin and subsequently eluting the diamine from the resin, such as CN109942437(A), CN110563594(A), CN110143882(A), CN108276293(A), U.S. Pat. No. 9,878,321(B2), U.S. Pat. No. 10,576,467(B2), U.S. Pat. No. 9,617,202(B2), Howel and Byus, Analytical Biochemistry (2002) 311(2), 127-32, and Lin et al. Chinese Journal of Chromatography (2018) 36(11), each of which is incorporated herein by reference in its entirety. 1189-1193. U.S. Patent No. 10,265,642(B2) and U.S. Patent No. 10,343,084(B2), each of which is incorporated herein by reference in its entirety, disclose a method for separating at least one amine selected from diamines and omega-amino acids from a feed mixture using simulated moving bed (SMB) adsorption technology. It further discloses that useful adsorbents are activated carbon, phlorizin, diatomaceous earth, molecular sieves, alumina, silica, silica-alumina, titania, polymeric resins containing one or more groups selected from sulfonate, hydroxy, amino, halogen, pyridyl, monosubstituted amino, unsubstituted amino, acyl, acyloxy, keto, alkoxy, and polymeric resins containing immobilized silver or lead, commonly known as immobilized metal affinity columns. Other adsorbents mentioned are Orpheus silica-based stationary phase adsorbents and Amberlite XAD-4, XAD-7, XAD-8 and XAD-418 resins, non-polar resins.The eluting solvent may be selected from water, diols, esters, nitriles, ketones, ethers, methanol, diols, esters and methyl ethers, aliphatic and cyclic ethers such as tetrahydrofuran and dioxane. Summary of the Invention [Problem to be solved by the invention]

[0004] Even though all the above methods have met with some success, they are limited by either expensive materials, generation of by-products-waste, suboptimal yields, or a combination thereof. Thus, there is a need for a simpler and more efficient method for separating diamines from reaction mixtures or fermentation broths containing amino acids and / or carboxylic acids at low cost and with optimal diamine yields without the need to use organic solvents. [Means for solving the problem]

[0005] Summary of the Invention A first aspect of the invention is a method of forming 1,5-pentanediamine (PDA), comprising contacting a mixture containing at least a 0.3 M solution of a lysine salt with sulfate or phosphate as a conjugate anion with an enzyme having lysine decarboxylase activity for a time sufficient to convert at least 50% of the lysine to PDA. In an exemplary embodiment, the lysine decarboxylase can be encoded by the cadA gene of E. coli.

[0006] In a preferred embodiment, the conjugate anion is a phosphate anion. In a preferred embodiment, the mixture is at a pH of 8.0-9.0. In a more preferred embodiment, the mixture contains 0.5-2.9 M lysine phosphate at a pH of 8.0-9.0. In a most preferred embodiment, the pH is 8.0-8.5. In the best practice of the above embodiment, at least 90% of the lysine is converted to PDA. In an illustrative example, the lysine salt is formed by adding sulfuric acid, more preferably phosphoric acid, to lysine free base in an amount sufficient to adjust the mixture to the desired pH.

[0007] The above embodiment may further comprise separating the PDA from the lysine and conjugated anions by contacting the reaction mixture with a strong base ion exchange resin and eluting the PDA from the resin, the eluted PDA being at least 85% pure with less than 15% lysine or conjugated anions. In a preferred embodiment, the strong base ion exchange resin is configured in a simulated moving bed apparatus.

[0008] A second aspect of the invention relates to a process for separating a diamine from an aqueous feedstock containing the diamine and at least one salt of an amino acid or a carboxylic acid, comprising contacting the aqueous feedstock at a pH in the range of 5.0 to 10.0 with a bed of a strong anion exchange resin, wherein the diamine has a purity of at least 85% and is eluted from the resin in a fraction containing less than 10% (w / w) of the amino acid or carboxylic acid.

[0009] In some embodiments, the diamine is 1,2-ethylenediamine, 1,3-propanediamine, 1,4-butanediamine (putrescine), 1,5-pentanediamine (cadaverine), 1,6-hexanediamine, 1,7-heptanediamine, and the like.

[0010] In one embodiment, the diamine is 1,5-pentanediamine (PDA) and the at least one amino acid salt is a lysine salt.

[0011] In one embodiment, the feedstock is obtained by decarboxylation of lysine by the action of lysine decarboxylase.

[0012] In another embodiment, the lysine salt is selected from the group consisting of lysine phosphate and lysine sulfate.

[0013] In another embodiment, less than 10% of the sulfate or phosphate conjugate anion of the lysine salt is present in the elution fractions containing the diamine.

[0014] In another preferred embodiment, the lysine salt is lysine phosphate.

[0015] In some other embodiments, the strong anion exchange resin comprises a quaternary ammonium salt.

[0016] In another more preferred embodiment, the purity of the PDA in the elution fraction is at least 95% and contains less than 5% (w / v) lysine.

[0017] In other embodiments, at least one amino acid and / or carboxylic acid is eluted from the anion exchange bed using a hydroxide salt.

[0018] In some embodiments, the hydroxide salt is ammonium hydroxide.

[0019] In another embodiment, the hydroxide solution is sodium hydroxide or potassium hydroxide.

[0020] In another embodiment, the feedstock has a pH in the range of 8.0 to 9.0.

[0021] In another embodiment, the beds are packed into a set of columns contained in a simulated moving bed apparatus comprised of a feed loading zone, a raffinate elution zone and a regeneration zone, where (a) the feed is loaded onto a first column segment defining the loading zone, (b) 1,5-pentanediamine is eluted from a second column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segments, (c) lysine and conjugated anions of salts of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the columns are regenerated in a regeneration zone, where the raffinate elution zone is not in fluid communication with the feed loading zone and where the regeneration zone is not in fluid communication with the raffinate elution zone.

[0022] In a preferred embodiment, a water wash is introduced into the column in a segment upstream of the first column segment defining the feed loading zone, a hydroxide salt solution is introduced into the column in a segment within the raffinate elution zone, and water is introduced into the column segment in the regeneration zone.

[0023] In another embodiment, the lysine salt is lysine phosphate.

[0024] In another preferred embodiment, the simulated moving bed apparatus comprises 12 column segments, of which the feed loading zone comprises 5 column segments, the raffinate elution zone comprises 3 column segments, and the regeneration zone comprises 4 column segments.

[0025] A preferred embodiment, the lysine salt is lysine phosphate.

[0026] A second aspect of the present invention relates to a method for producing a diamine, in one embodiment, the method comprises: (a) culturing a host microbial organism engineered to produce a diamine in a medium that includes a source of phosphate or sulfate to form a phosphate or sulfate; (b) treating the culture of a microorganism to produce a feedstock comprising a diamine; (c) contacting the feedstock with a bed of a strong anion exchange resin in a column; (e) eluting the diamine from the column in a first fraction separate from a second fraction containing amino and carboxylate salts; wherein the diamine is eluted from the column with a purity of at least 95% and contains less than 5% amino acids and / or carboxylic acids.

[0027] In some embodiments, the diamine is selected from 1,3-propanediamine, 1,4-butane-diamine (putrescine), 1,5-pentanediamine (cadaverine), 1,6-hexanediamine, and 1,7-heptanediamine.

[0028] In some preferred embodiments, the column comprises a set of columns housed in a simulated moving bed apparatus consisting of a feed loading zone, a raffinate elution zone and a regeneration zone, where (a) the feed is loaded onto a first column segment defining the feed loading zone, (b) the diamine is eluted from a second column segment downstream from the feed loading zone with respect to the direction of liquid flow through the column segments, (c) the lysine and conjugated anions of a salt of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the column is regenerated in the regeneration zone, where the raffinate elution zone is not in fluid communication with the feed loading zone and where the regeneration zone is not in fluid communication with the raffinate elution zone.

[0029] In another embodiment, the method is a method for producing 1,5-pentanediamine (PDA), comprising: (a) obtaining a culture broth resulting from fermenting a host microorganism engineered to produce lysine; (b) removing the microorganisms from the culture broth; and (c) adding a phosphate or sulfate source to the broth to form a phosphate or sulfate salt of lysine; (d) contacting the culture broth containing sulfate or phosphate with lysine decarboxylase to decarboxylate lysine and form a culture broth containing lysine salt and PDA; (e) contacting the culture broth containing the lysine salt and the PDA with a bed of a strong anion exchange resin in a column; (f) eluting the PDA from the column in a first fraction separate from a second fraction containing lysine and lysine salts; wherein the PDA is eluted from the column with a purity of at least 95% and contains less than 5% lysine.

[0030] In a preferred embodiment, the lysine salt is lysine phosphate.

[0031] In a more preferred embodiment, the column comprises a set of columns housed in a simulated moving bed apparatus consisting of a feed loading zone, a raffinate elution zone and a regeneration zone, where (a) the feed is loaded onto a first column segment defining the feed loading zone, (b) 1,5-pentanediamine is eluted from a second column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segments, (c) lysine and conjugated anions of salts of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the column is regenerated in the regeneration zone, where the raffinate elution zone is not in fluid communication with the feed loading zone and where the regeneration zone is not in fluid communication with the raffinate elution zone.

[0032] Brief explanation of the figure The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 1 shows a pulse test comparison eluting 1,5-pentanediamine (cadaverine) from DOW 22™, PA208™, and HPA 25 L™ using deionized water as the eluent. [Figure 2B] FIG. 1 shows a pulse study comparing the effect of pH on retention of 1,5-pentanediamine from DOW22™, PA208™, and HPA 25 L™. [Diagram 2] FIG. 1 shows a pulse study showing the elution profile and purity of 1,5-pentanediamine from a column packed with DOWEX 22™ anion exchange resin. [Diagram 3] 1 shows a pulse study showing the elution profile and purity of 1,5-pentanediamine from a column packed with PA308™ anion exchange resin. [Figure 4]FIG. 1 shows a pulse test showing the elution profile and purity of 1,5-pentanediamine from a column packed with HPA25L™ anion exchange resin. [Diagram 5] FIG. 1 shows the column elution profile for the separation of 1,5-pentanediamine from phosphate and lysine on PA 209 strong base ion resin. [Figure 6] FIG. 1 shows a schematic diagram of a simulated moving bed chromatography system for purifying diamines from a complex reaction mixture. [Figure 7A] For comparative purposes, the conversion of lysine to 1,5-pentanediamine (also known as PDA or cadaverine) by E. coli lysine decarboxylase over time is shown, using lysine hydrochloride as the lysine salt. [Figure 7B] 1 shows a time course of lysine conversion using lysine sulfate as the lysine salt according to one embodiment of the present invention. [Figure 7C] 1 shows a time course of lysine conversion using lysine phosphate as a lysine salt according to another embodiment of the present invention. [Figure 8] 4 shows the percent conversion of lysine to 1,5-pentanediamine at various pH values ​​using a 1 M solution of lysine free base adjusted with phosphoric acid to the indicated pH. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description Some aspects of the present invention are described herein in connection with the application of examples for illustration. It should be understood that many specific details, relationships, and methods are described to provide a detailed understanding of the features described herein. However, a person skilled in the relevant art will readily recognize that the features described herein can be implemented without one or more of the specific details or by using other methods. Unless otherwise specified, the features described herein are not limited by the illustrated order of acts or events, since some acts may be performed in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to carry out a methodology in accordance with the features described herein.

[0035] The terminology used herein is for the purpose of describing exemplary embodiments only and is not intended to be limiting. The singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent that "including," "comprises," "has," "having," "comprising," or variations thereof are used anywhere in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to "including."

[0036] In this disclosure, the term "about" or "approximately" means within a range of 10%, preferably 5%, and more preferably 3% of a given value. In this disclosure, the term "substantially" means something that can occur to a greater extent or degree.

[0037] The term "diamine" as used herein means any organic compound containing two or more amino groups. Examples of diamines of the present invention are, but are not limited to, 1,2-ethylenediamine (also known as diaminoethane), 1,3-propanediamine (also known as trimethylenediamine), 1,4-butanediamine (also known as putrescine or tetramethylenediamine), 1,5-pentanediamine (also known as cadaverine or pentamethylenediamine, PDA or PMDA), 1,6-hexanediamine (also known as hexamethylenediamine), 1,7-heptanediamine (also known as heptamethylenediamine) and its isomers, derivatives and analogs, and aromatic amines, such as, but not limited to, aliphatic diamines, such as o-, m- or p-phenylenediamine, 4,4'-diaminobiphenyl and its isomers, derivatives and analogs. It should be noted that some diamines are known by several acceptable names in the art, including, but not limited to, the names listed above.

[0038] As used herein, the term "fermentation" refers to the process of producing compounds, such as diamines, organic acids, amino acids, or enzymes, such as lysine decarboxylase, by growing wild-type or engineered microorganisms that produce the compound or enzyme of interest.

[0039] The term "fermentation broth" as used herein means, without limitation, a liquid medium in which a microorganism converts an organic carbon source into an organic material of interest, such as a diamine, amino acid, or amino acid decarboxylase, and may contain other organic materials, such as carboxylic acids and alcohols, as by-products, and typically also contains nutrients and salts required for the growth of the organism, pH control, or to form salts of compounds produced by fermentation. The term encompasses whole broths containing the microorganism of interest and clarified broths where the microorganism has been separated from the remaining components.

[0040] The term "carbon source" as used herein refers to a carbon-containing nutrient required for microbial growth and production of a product of interest. Carbon sources include, but are not limited to, carbohydrates such as glucose, fructose, sucrose and starch, tryptone, carboxylic acids and / or salts thereof, such as, but not limited to, acetic acid, tartaric acid, citric acid, amino acids and / or salts thereof, and triglycerides.

[0041] The term "bed volume" or "column volume" means the volume of liquid in a packed column or bed.

[0042] A first aspect of the present disclosure is the discovery that lysine sulfate and lysine phosphate, particularly lysine phosphate, are superior to lysine hydrochloride as substrates for the conversion of lysine to PDA using lysine decarboxylase in terms of achieving high conversion rates at low enzyme dosages. Furthermore, lysine sulfate and lysine phosphate can be used at much higher concentrations than HCl, making reactions with these substrates more suitable for industrial-scale production. Furthermore, the use of lysine sulfate or lysine phosphate allows the reaction with lysine decarboxylase to proceed at high efficiency over a wide pH range of 5.0 to 9.0.

[0043] The enzymatic conversion of lysine to PDA by a lysine decarboxylase called CadA, encoded by the CadA gene from Escherichia coli (E. coli), is a known reaction. CadA is a pyridoxal 5-phosphate-dependent lysine decarboxylase that is induced by lysine in acidic systems. The natural useful pH range of the enzyme is 5-7, with a recorded optimum pH value of 5.5. It is known that the enzyme dissociates and forms a less active dimer if the pH is increased above 7 (Kou et al. Characterization of a new lysine decarboxylase from Aliivibrio salmonicida for cadaverine production at alkaline pH, Journal of Molecular Catalysis B: Enzymatic, vol 133, Supp. 1, 588-594, 2016). The literature shows a sharp decrease in activity at pH above 5.5 (Kou et al 2016). Lysine free base has a pH of 10.2, which is far too high for CadA to function, and therefore acidic (anionic) salts of lysine must be used to lower the pH. However, previous studies performed with CadA for lysine decarboxylation have exclusively used lysine hydrochloride as a substrate, with some using hydrochloric acid to maintain the pH during the reaction. Alternative lysine salts, such as lysine sulfate or lysine phosphate, have not been evaluated.

[0044] From this disclosure it has become clear that high conversion of lysine to PDA with lysine decarboxylase, exemplified by CadA, can occur when lysine sulfate, particularly lysine phosphate, is used at concentrations higher than 0.5M up to the solubility limit of lysine phosphate, which is about 3.0M. In contrast, the use of lysine hydrochloride is limited by its solubility at higher concentrations. A 2.75M lysine hydrochloride solution, pH adjusted to 5.5, is insoluble at the temperature at which CadA is active. The use of lysine phosphate or lysine sulfate increases the solubility and allows the reaction to proceed at the optimum CadA temperature of 37°C at a much higher conversion rate than is possible with lysine hydrochloride. Furthermore, the use of these salts, particularly lysine phosphate, can extend the pH range of the CadA enzyme to as high as 9.0, where dissociation of the enzyme would normally occur when lysine hydrochloride is used.

[0045] To evaluate the effect of conjugate anion salts of lysine on the conversion of lysine to PDA using lysine decarboxylase, an E. coli strain designated BB16.9.8 was engineered to overexpress CadA. BB16.9.8 contains a copy of T7 polymerase under the control of the lac promoter, which is inducible by lactose or IPTG. A copy of the E. coli lysine decarboxylase cadA gene under the control of the T7 promoter was also integrated into the genome adjacent to the thrC gene. The strain was grown to logarithmic phase in the presence of an inducing amount of lactose to overexpress CadA, then rapidly cooled and centrifuged to obtain a cell paste. The cell paste was lysed using BUG BUSTER™ cytochemical lysis medium, which rendered the cells porous and formed a crude homogenate, which was centrifuged to obtain a clarified crude extract with a protein concentration of approximately 4.0 mg / ml, referred to herein as the CadA lysate. One molar solutions of lysine hydrochloride, lysine sulfate, and lysine phosphate were prepared by adding hydrochloric acid, sulfuric acid, or phosphoric acid in molar equivalents to a 1 M solution of lysine free base in an amount sufficient to adjust the pH to the desired level. These samples were evaluated for the ability of CadA lysate to convert lysine to PDA using an assay mixture containing pyridoxal phosphate, a necessary cofactor for lysine decarboxylase. Exemplary reactions were performed in 10 milliliters to 30 liters of lysine salt solution, and the amount of CadA lysate added was varied in different experiments.

[0046] In a first illustrative experiment with 250 g of 1 M lysine hydrochloride (pH 5.5), 37 mg of CadA lysate was incubated with that amount of lysine hydrochloride for 24 hours, and time points were taken every hour for the first 5 hours and at 24 hours. The reaction at selected time points was stopped by heating at 70° C. for 1 hour. The conversion of lysine to PDA was assessed by HPLC. FIG. 7A shows that the reaction proceeded linearly over the first 3 hours, by which time about 50% of the lysine had been converted to PDA, and then slowed, resulting in only about 70% conversion at the 24 hour time point.

[0047] In other experiments using 1M lysine sulfate and 1M lysine phosphate adjusted to pH 5.5, various amounts of CadA (measured by total protein) were added to the reaction mixture and time points were taken over a much shorter reaction period of only 4 hours, as we have previously shown that reactions with these salts are much faster than with lysine hydrochloride. From Figure 7B it can be seen that for lysine sulfate, using only 5 mg of crude extract protein in a 10 ml reaction, approximately 60% conversion to PDA occurred, reaching approximately 70% conversion at 6 hours. Figure 7C shows a more surprising result, using only lysine phosphate and 5 mg of protein, over 90% of the lysine was converted to PDA after 2 hours, with over 95% conversion after 6 hours.

[0048] Another advantage of using lysine sulfate or, more preferably, lysine phosphate for the conversion of lysine to PDA by lysine decarboxylase is that it broadens the useful pH range of the enzyme. As described herein above, E. coli CadA lysine decarboxylase has an optimum pH value of 5.5 and rapidly loses activity at pHs above 7.0. In contrast, the inventors have discovered that using lysine sulfate or lysine phosphate, particularly lysine phosphate, allows the enzyme to remain active up to at least pH 9.0. Most methods for producing lysine by fermentation produce lysine in a free base form with a pH of about 10.2, which is well above the useful pH range of E. coli lysine decarboxylase. The use of lysine sulfate or, more preferably, phosphate broadens the useful pH range of the enzyme, thereby allowing the use of less acid (sulfuric acid or phosphoric acid) that provides the conjugate anion to the lysine salt than would be required to form lysine hydrochloride at the pH range in which the CadA enzyme is active, thereby reducing the amount of anion that must be separated from the PDA. Figure 8 shows that at pH 5.5-8.0, at least 84% of the lysine in a 1 M solution of lysine phosphate can be converted to PDA in 24 h using only 5 mg of CadA lysate in a 10 ml reaction. A conversion rate of at least 97% was observed at pH 5.5-8.5.

[0049] Another advantage of using lysine sulfate or most preferably lysine phosphate is the ability to achieve much higher concentrations than can be achieved with lysine hydrochloride. Examples 1-9 show high conversion rates after 24 hours of reaction using CadA in reactions containing 0.57-2.86M lysine adjusted to various pH values ​​with phosphoric acid. Thus, another feature of the invention is the conversion of lysine to PDA to form lysine phosphate in reactions containing lysine decarboxylase and at least 0.3M lysine, adjusted to a pH of 8.0-9.0. In an exemplary embodiment, the mixture contains 0.5M-2.9M lysine and is adjusted to a pH of 8.0-9.0. More preferably, the lysine concentration is 1.0M-2.5M or more preferably 1.5M-2.25M and the pH is 8.0-8.5. In a most preferred embodiment, the lysine concentration is 1.75-2.25M and the pH is 8.5. Reaction times and enzyme amounts can be varied to achieve at least 50%, at least 70%, at least 80%, at least 90% and most preferably at least 95% conversion of lysine to PDA, with some reactions achieving at least 98% conversion.

[0050] A second aspect of the present invention relates to a process for separating PDA from lysine and the conjugated anions of lysine salts. The present invention is applicable to the separation of diamines from an aqueous feedstock containing diamines and at least one salt of an amino acid and / or a carboxylic acid. This aspect of the invention comprises contacting an aqueous feedstock at a pH in the range of 5.0-10.0, preferably 6.0-9.5, more preferably 6.5-9.0, more preferably 7.0-9.0, more preferably 8.0-9.0 and most preferably about 8.5 with a bed of strong anion exchange resin, wherein the diamine has a purity of at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% (w / w) and is first eluted from the resin containing less than 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% (w / w) of amino acid or carboxylic acid, and the amino acid salt, and / or carboxylate salt, and / or conjugate ions of the amino acid salt are separately eluted with a hydroxide salt solution. The feedstock is an aqueous solution, typically a clarified fermentation broth, containing at least the diamine product and one or more reagents that will react to produce the diamine, including but not limited to unreacted raw organic and inorganic salts, such as amino and carboxylic acids and their salts, dicarboxylic acids and their salts, tricarboxylic acids and their salts, pyridoxal 5'-phosphate, inorganic salts, including but not limited to alkali and alkaline earth metal salts such as phosphates, sulfates, chlorides, and other metabolic products, including but not limited to ethanol, propanol, and by-products produced by any chemical or biochemical process to produce the diamine, found in the medium. The feedstock may contain less than 10% (w / w), preferably less than 8% (w / w), preferably less than 6% (w / w), preferably less than 5% (w / w) of alcohol, such as ethanol or propanol, produced by the fermentation process. The amino acids and / or carboxylic acids are eluted from the resin using an aqueous solution of a hydroxide salt, such as, but not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, at a concentration in the range of 0.5-10% (w / v), preferably 1-9% (w / v), more preferably 2-8% (w / v), more preferably 3-7% (w / v), and most preferably 4-6% (w / v).

[0051] The process of the present invention is particularly suitable for separating diamines from a reaction mixture or fermentation broth feedstock containing amino acids and / or carboxylic acids. In some preferred embodiments, the diamine is selected from the group consisting of 1,2-ethylenediamine, 1,3-propanediamine, 1,4-butanediamine (putrescine), 1,5-pentanediamine (cadaverine), 1,6-hexanediamine and 1,7-heptanediamine. In one preferred embodiment, the diamine is 1,5-pentanediamine. One of the many advantages of this method is that the diamine is an early eluting fraction from the resin, resulting in substantially improved product recovery, and later eluting products such as amino acid salts, conjugate ions of amino acid salts and other inorganic salts can be recovered and recycled to the fermentation process to produce diamines or converted to other useful products.

[0052] Strong anion exchange resins are generally polymer matrices that contain quaternary ammonium groups. Standard commercially available strong anion exchange resins are -N + (CH3)3 (Type 1 resin) or -N + (CH3)2(C2H4OH) (type 2 resin). The separation of chemical compounds on ion exchange resins depends on the degree of interaction between the charged groups of the stationary phase and the molecules in the mobile phase. For example, strong ion exchange resins containing quaternary ammonium cations will interact strongly with negatively charged molecules and will preferentially retain negatively charged molecules, while neutral and positively charged molecules will interact less readily with the resin and will more preferentially flow with the mobile phase. Examples of commercially available strong anion exchange resins and their manufacturers are listed in Table 1. In some preferred embodiments, the anion exchange resin is selected from DOW 22™, HPA 25L™, and Mitsubishi PA DIAION PA308™. It is a porous resin containing trimethylammonium cations and efficiently separates diamines from mixtures of diamines with amino acid salts and / or carboxylate salts at high flow rates.

[0053] [Table 1]

[0054] As mentioned above, the mixture applied to the resin can be obtained from a chemical or biochemical process that produces a diamine or an amino acid that is converted to a diamine. In a preferred embodiment, the feedstock is a clarified fermentation broth obtained from the fermentation of a microorganism to produce the diamine or amino acid of interest. In an embodiment illustrated by the examples herein, the clarified fermentation broth is obtained by fermenting bacteria to produce lysine and removing the bacteria by filtration to produce a clarified fermentation broth containing lysine free base. A mineral acid or a mineral salt of phosphoric acid or sulfuric acid, exemplified by sulfuric acid or phosphoric acid, is added to the clarified broth to form lysine sulfate and lower the pH to a level at which lysine decarboxylase is active. The mixture is pH adjusted to be in a range that is optimal for the functionality of lysine decarboxylase, which is added to the mixture for a time sufficient for the conversion of lysine to 1,5 pentanediamine (PDA). At the end of the reaction period, the mixture is applied to a strong anion exchange resin in a column which is eluted to obtain a first fraction which is at least 85%, more preferably at least 90% and even more preferably at least 95% PDA and contains less than 15%, less than 10% or more preferably less than 5% lysine plus conjugated sulfate or phosphate anions, followed by elution of the column with a hydroxide base to remove residual lysine and conjugated anions from the resin.

[0055] The present invention may be practiced using fermentation of microorganisms known in the prior art to produce diamines. Examples of microorganisms that produce diamines: U.S. Patent No. 10,711,289, the entirety of which is incorporated herein by reference, discloses microorganisms engineered to produce ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, 1,5-pentanediamine, 1,6-hexanediamine, and 1,7-heptanediamine. U.S. Patent No. 10,472,636, the entirety of which is incorporated herein by reference, discloses microorganisms engineered to produce 1,3-propanediamine and 1,5-pentanediamine (cadaverine). Nos. 11,124,812, 11,053,525, 11,053,524, and 10,870,871, each of which is incorporated herein by reference in its entirety, disclose one or more microorganisms, such as Corynebacterium glutamicum, Providencia rettgeri, Brevibacterium flavum, Brevibacterium lactofermentum, and Serratia marcescens, and Escherichia coli, that have been engineered to produce 1,4-butanediamine (putrescine). Nos. 8,741,623, 10,626,425, 10,640,798, 9,745,608, 9,644,220, 9,365,876, 9,115,362, and 8,741,623, each of which is incorporated by reference in its entirety, disclose one or more microorganisms engineered to produce 1,5-pentanediamine.Among the microorganisms disclosed for producing 1,5-pentanediamine are Corynebacterium glutamicum, Providencia rettgeri, Brevibacterium flavum, Brevibacterium lactofermentum, and Serratia marcescens, as well as Escherichia coli. No. 10,150,977, the entirety of which is incorporated herein by reference, describes Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas mobilis, and other strains engineered to produce 1,6-hexanediamine. mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens and Pseudomonas putida are disclosed.

[0056] The mixture applied to the column is obtained by filtering solids and / or removing the microorganisms from the broth to form a clarified fermentation broth. Furthermore, in some cases, the cells of the microorganisms may be lysed and the solids removed by filtration to generate the feedstock for the column or the feedstock for the reaction that will form the diamine that is applied onto the column. In some cases, it may also be desirable to remove proteins and nucleic acids from the feedstock before isolating the diamine by methods known in the art, such as, but not limited to, acidifying the culture broth to precipitate proteins and nucleic acids.

[0057] In some embodiments, the feedstock may be prepared from a medium containing a diamino acid, such as lysine or ornithine, using a microorganism engineered to express an enzyme with amino acid decarboxylase activity. For example, U.S. Patent No. 8,871,477, the entirety of which is incorporated herein by reference, discloses a method for producing cadaverine by culturing a transformed Corynebacterium glutamicum or E. coli engineered to express and secrete both lysine and lysine decarboxylase in the medium. In another example, U.S. Patent No. 11,155,840, the entirety of which is incorporated herein by reference, discloses a microorganism from a Bacillus sp., such as B. subtilis, licheniformis, engineered to produce lysine and a thermophilic lysine decarboxylase that produces PDA by culturing the Bacillus sp.

[0058] In yet another preferred embodiment, the feedstock for the column is obtained by in vitro decarboxylation of amino acids using purified or partially purified amino acid decarboxylases or cell extracts obtained from the culture of microorganisms producing said decarboxylases. For example, U.S. Patent No. 10,351,839, incorporated herein by reference, discloses microorganisms engineered to produce pH-stable lysine decarboxylases.

[0059] Simulated Moving Bed (SMB) The present invention is best carried out using a simulated moving bed (SMB) system. A SMB system suitable for separating at least one diamine from a feed mixture comprises several column segments forming a plurality of zones, each of which performs a chromatographic operation. For example, the SMB may comprise a first section of the column segment for separating the product component of interest, a second section for separating the second component, and one or more wash sections for regenerating the resin. Each section comprises a plurality of segments, each of which contains a bed of solid strong anion exchange resin. Each section further comprises one or more injection points for the feed mixture, one or more injection points for an eluent, for example comprising water or an aqueous solution of a hydroxide salt, a withdrawal point for an extract stream, and a withdrawal point for a raffinate stream. In most cases, the columns in each section are in fluid communication with each other, although certain sections may be separated from other sections for the purpose of applying a wash or regeneration step.

[0060] The SMB may be equipped with multiple valves attached to each column segment so that any feed stream may be introduced into any section or zone and any outlet or effluent stream may be removed from any section or zone. In certain embodiments, the multiple valves rotate as a rotating unit on the fixed column segment, stepping from one segment to an adjacent segment in a direction opposite to the direction of fluid flow-through through the SMB system, mimicking the effect that would occur if the solid phase was moving while the liquid phase was stationary. In other embodiments, the valves may remain stationary and the column segments move as a rotating unit under the stationary valves in a direction opposite to the fluid flow to provide the same effect.

[0061] During operation of the SMB, the inlet connection through which the feed stream is fed and the outlet connection through which the outlet stream is withdrawn are moved or indexed from each column to the adjacent column at regular intervals. For example, in one embodiment, the location of the inlet and outlet streams can be moved intermittently from a column to the next adjacent column in the opposite direction of the liquid eluent flow to achieve separation of at least one diamine from the amino acid and / or carboxylic acid. The intermittent movement of the inlets and outlets in the direction of the liquid eluent flow simulates the countercurrent movement of one or more beds of solid adsorbent. Different devices and operating methods can be used to simulate the countercurrent movement of solids against liquid. Known simulated or actual moving bed chromatography devices can be used for the purpose of separating diamines from amino acids and / or carboxylic acids and their salts from a feed mixture such as a fermentation product. As non-limiting examples, the apparatus described in U.S. Pat. No. 2,985,589, U.S. Pat. No. 3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533, FR-A-2103302, FR-A-2651148, FR-A-2651149, U.S. Pat. No. 6,979,402, U.S. Pat. No. 5,069,883 and U.S. Pat. No. 4,764,276, the entireties of which are incorporated herein by reference, may be constructed and operated in accordance with the present disclosure to separate amino acids and / or carboxylic acids and their salts, as well as diamines from inorganic salts such as, but not limited to, phosphates and / or sulfates, from a feed mixture such as a fermentation product.

[0062] In an exemplary process for separating diamines from amino acids and carboxylic acids and their salts, strong anion exchange resins, such as, but not limited to, Mitsubishi PA DIAION PA308™, DOW22™, and HPA 25L™, are packed onto a set of columns housed in a simulated moving bed apparatus consisting of a feed loading zone, a raffinate elution zone, and a regeneration zone (see, for example, FIG. 5). The feed is loaded onto a first column segment that defines the feed loading zone. The diamine is eluted from another column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segment. Conjugated anions and inorganic salts of amino acids and / or carboxylic acids and salts of amino acids and / or carboxylic acids are eluted from yet another column in the raffinate elution zone. The column is regenerated in the regeneration zone. In such an arrangement, the raffinate elution zone is not in fluid communication with the feed loading zone, and the regeneration zone is not in fluid communication with the raffinate elution zone. In a preferred embodiment, the water wash is introduced into the column in a segment upstream of the first column segment defining the feed loading zone, the hydroxide salt solution is introduced into the column in a segment within the raffinate elution zone, and water is introduced into the column segment in the regeneration zone (see FIG. 6). EXAMPLES

[0063] Working Example In the following examples, the concentration of lysine refers to the concentration of lysine calculated as the lysine free base concentration before the anionic acid is added to form the lysine salt. Unless otherwise specified, 10 ml of lysine salt solution was used in the reaction in Examples 1 to 9.

[0064] Example 1 Conversion of 9.7% Lysine Sulfate to 1,5-Pentanediamine (PDA) at pH 5.6 100.52 g of a 9.7 wt% lysine sulfate solution (0.66M lysine) was pH adjusted to 5.6 with sulfuric acid and pre-heated to 37°C with 1.80 mg of pyridoxal 5-phosphate in a 100 ml reaction to which 4.0 ml of cell-free crude lysate containing E. coli lysine decarboxylase (CadA) with a protein concentration of approximately 12 mg / ml was added. The mixture was gently stirred for 24 hours and sampled periodically. The reaction reached 71% conversion of lysine to cadaverine within 3 hours, with 98% lysine conversion at 24 hours indicating that lysine sulfate can be used as a suitable substrate for CadA.

[0065] Example 2 Conversion of 8.4% Lysine Phosphate to PDA at pH 5.5 300.98 g of 8.4 wt % lysine phosphate solution (0.57 M lysine) was pH adjusted to 5.5 with phosphoric acid to give a final volume of approximately 290 ml and pre-heated to 37° C. 146.3 mg of protein from cell-free crude lysate containing E. coli lysine decarboxylase was added and 5.08 mg of pyridoxal 5-phosphate was added. The mixture was gently stirred for 24 hours and samples were drawn throughout the reaction. Final conversion was achieved in 3 hours with a lysine conversion of 98%. The results indicate that lysine phosphate can be used as a suitable substrate for CadA.

[0066] Example 3 Conversion of 13.5% Lysine Phosphate to PDA at pH 8.0 100.93 g of 13.5 wt % lysine phosphate solution (0.92 M lysine) was pH adjusted to 7.958 with phosphoric acid and 49.4 mg of protein from the cell-free crude lysate was added along with 1.60 mg of pyridoxal 5-phosphate. The mixture was heated to 37° C. and gently stirred for 24 h. The sample was quenched, resulting in 96% conversion in 100 min, with a final conversion of 98% lysine achieved within 3 h. The results indicate that CadA can function at pH 8.0 with high efficiency using lysine phosphate.

[0067] Example 4 Conversion of 37.1% lysine phosphate to PDA at pH 7.7 98.09 g of 37.1 wt % lysine phosphate solution (2.54 M lysine) was adjusted to pH 7.727 with phosphoric acid to give a 98 ml sample to which 150.17 mg of protein from the cell-free crude lysate was added along with 1.70 mg of pyridoxal 5-phosphate. The mixture was heated to 37° C. and gently stirred for 24 hours. The sample was quenched and a final conversion of 70% lysine was achieved by 24 hours.

[0068] Example 5 Conversion of 41.8% lysine phosphate to PDA at pH 7.9 109.01 g of 41.8 wt% lysine phosphate solution (2.86 M lysine) was adjusted to pH 7.91 with phosphoric acid and 174.96 mg of protein from the cell-free crude lysate was added along with 1.70 mg of pyridoxal 5-phosphate. The mixture was heated to 37° C. and gently stirred for 24 hours. The sample was quenched and a final conversion of 48% lysine was achieved by 24 hours.

[0069] Example 6 Conversion of 33.6% lysine phosphate to PDA at pH 8.4 10 mL of 33.6 wt% lysine phosphate solution (2.30 M lysine) was adjusted to pH 8.4 with phosphoric acid and 5.6 mg pyridoxal 5 phosphate. The feed was split 4 ways to dose the crude cell extract. Two tubes received low amounts of protein, 119 mg and 127 mg, and two tubes received higher amounts, 256 and 262 mg. The control contained no enzyme added. All samples were incubated at 37°C. The low dose samples showed lysine conversions of 30.4 and 39.6% at 24 hours. The high dose samples had lysine conversions of 79.5% and 83.4% at 24 hours.

[0070] Example 7 Conversion of 17.9% lysine phosphate to PDA at pH 8.5 100.57 g of 17.9 wt% lysine phosphate solution (1.22 M lysine) was adjusted to pH 8.469 with phosphoric acid and 49.84 mg of protein was added along with 1.70 mg of pyridoxal 5-phosphate from the cell-free crude lysate. The mixture was heated to 37° C. and gently stirred for 24 hours. The sample was quenched and a final conversion of 95% lysine was achieved within 3 hours and 97% by 24 hours.

[0071] Example 8 Conversion of 16.8% lysine phosphate to PDA at pH 8.4 A 16.8 wt% lysine phosphate solution (1.15M lysine) was prepared to which 2 liters of CadA crude extract was added along with 507.1 mg of pyridoxal 5-phosphate in a final reaction volume of 29072.67 mL. The final pH was 8.39. The reaction was stirred gently at 37°C and samples were taken at regular intervals. A conversion of 95% was obtained in the first hour and 99.5% was achieved by 24 hours.

[0072] Example 9 Conversion of 18.9% lysine phosphate to PDA at pH 9.0 100.58 g of 18.9 wt % lysine phosphate solution (1.29 M lysine) was adjusted to pH 8.949 with phosphoric acid and 49.84 mg of protein from the cell-free crude lysate along with 1.70 mg of pyridoxal 5-phosphate was added and heated to 37° C. The mixture was gently stirred for 24 h and the sample was quenched; a final conversion of 63.5% lysine was achieved within 3 h and 85.2% by 24 h.

[0073] Example 10 Separation of 1,5-pentanediamine from the feedstock: (a) Feedstock: The feedstock was a mixture resulting from the decarboxylation of lysine by the action of zymide decarboxylase after the formation of lysine phosphate by adding sufficient phosphoric acid to the lysine base to adjust the pH to 8.5, as described in Example 8. The feedstock contained 90 g / L 1,5-pentanediamine, 0.27 g / L lysine, and 40 g / L phosphate.

[0074] (b) Preparation of a strong anion exchange column: A slurry of each of Dowex22™, PA308™ and HPA25L™ strong anion exchange resins (100 mL) in deionized water was loaded into a jacketed glass column. Air bubbles were removed from the resin bed and rinsed with 3 column volumes of degassed deionized water at a flow rate of 3 column volumes / hr. The packed column was conditioned by pumping 5 column volumes of 4% sodium hydroxide solution at a flow rate of 5 column volumes / hr, followed by degassed deionized water at the same flow rate, until the effluent pH stabilized. The liquid level in the column was lowered to level with the top of the resin bed.

[0075] (c) Purification of 1,5-pentanediamine: Approximately 0.8-1.0 column volumes of the feedstock were loaded onto the three columns and 1,5-pentanediamine was eluted from the columns at 40 °C with degassed deionized water at a flow rate of 5 mL / min, followed by lysine and phosphate elution with 4% (w / w) aqueous sodium hydroxide. Fraction 1 of 8 mL (0.08 column volumes) was collected and analyzed for content to determine the purity of 1,5-pentanediamine in each fraction. Figure 3A shows a comparative study of eluting 1,5-pentanediamine (cadaverine) from anion exchange columns packed with DOW22, PA208 and HPA25. Figures 2-4 show the elution profile and purity of 1,5-pentanediamine and lysine content in each fraction. As shown in Figures 2-4, the diamine fractions eluted from the columns were greater than 99% pure.

[0076] Example 11 Effect of pH on the recovery of 1,5-pentanediamine: To test the effect of pH on the elution profile and yield, the pH of the feed was adjusted to the desired pH value using sodium hydroxide and sulfuric acid. Figure 1B shows the effect of feed pH on the elution profile of 1,5-pentanediamine from three columns packed with DOW22, PA308 and HPA25L.

[0077] Example 12 Single-column purification of 1,5-pentanediamine on PA308 resin The feed material was a lysine enzymatic decarboxylation reaction mixture containing 90 g / L PDA, 0.27 g / L lysine and 40 g / L phosphate, adjusted to pH 8.5 with phosphoric acid as described in Example 8. 0.8 column volumes (BV) of the feed material was applied onto a 100 ml column (15 mm x 600 mm) and then eluted with water at two different feed rates, followed by 4% NaOH at the feed rates shown below. Fraction collection: Fractions were collected every 0.08 BV (8 mL). Column temperature: 40℃ Feed rate (0.0~0.8BV): 32mL / min Elution 1 rate (0.8~1.8BV): 32mL / min Elution 2 speed (1.8~4.8BV): 5mL / min Eluent 1 (0.8-1.8 BV): Degassed deionized water Eluent 2 (1.8-4.8BV): 4% NaOH

[0078] As can be seen from Figure 5, the PDA (cadaverine) product was recovered in high purity after 0.3-1.0 BV in the initial feed loading and water wash (wash 1) fractions, after which lysine and phosphate were found in the eluent. Following this, the column was regenerated by eluting with 4% NaOH to remove adsorbed phosphate and residual cadaverine and lysine. Cadaverine recovered in product fractions up to 1 BV was 100% pure.

[0079] Example 13 Purification of 1,5-pentanediamine using simulated moving bed: Figure 6 shows a schematic diagram of a simulated moving bed chromatography system designed to produce diamines from a complex reaction mixture. Each of 12 column segments of 450 ml was packed with strong base anion exchange resin PA308. A feed sample with the composition shown in Table 2 was obtained as described in Example 12 and was continuously loaded onto the resin at 1.15 liters / hour. It was loaded onto column 10 in adsorption zone I and the 1,5-pentanediamine product was continuously recovered from column 12. In zone II the column was continuously washed with water. In zone III, the desorption zone, the negatively charged ions and residual lysine were continuously eluted from the column with 4% sodium hydroxide solution. Samples of the product diamine, eluents from the desorption zone, first (raffinate) and wash 2 (sodium hydroxide eluent) were collected and analyzed and their compositions are shown in Table 2.

[0080] [Table 2]

Claims

1. 1. A process for separating a diamine from an aqueous feedstock containing said diamine and at least one conjugated anion salt of an amino acid or a carboxylic acid, comprising contacting said aqueous feedstock at a pH in the range of 5.0 to 10.0 with a bed of a strong anion exchange resin; and eluting said diamine from said resin in a fraction having a purity of at least 85% and containing less than 10% (w / w) of said amino acid or carboxylic acid and conjugated anion.

2. 2. The process of claim 1, wherein the diamine is 1,5-pentanediamine (PDA) and the at least one amino acid salt is a lysine salt.

3. 2. The process of claim 1, wherein the feedstock is obtained by decarboxylation of lysine by the action of lysine decarboxylase.

4. 4. The process of claim 3, wherein the lysine salt is selected from the group consisting of lysine phosphate and lysine sulfate.

5. 5. The process of claim 4, wherein less than 10% of the sulfate or phosphate conjugate anion of the lysine salt is present in the elution fraction containing the diamine.

6. 6. The process of claim 5, wherein the lysine salt is lysine phosphate.

7. 3. The process of claim 2, wherein the strong anion exchange resin comprises a quaternary ammonium salt.

8. 3. The process of claim 2, wherein the purity of PDA in the elution fraction is at least 95% and contains less than 5% (w / v) lysine.

9. 10. The process of claim 1, wherein the at least one amino acid and / or carboxylic acid is eluted from the anion exchange bed using a hydroxide salt.

10. 3. The process of claim 2, wherein the lysine is eluted from the anion exchange bed using a hydroxide salt.

11. 11. The process of claim 10, wherein the hydroxide salt is ammonium hydroxide.

12. 11. The process of claim 10, wherein the hydroxide solution is sodium hydroxide.

13. 10. The process of claim 1, wherein the feedstock has a pH in the range of 8.0 to 9.

0.

14. 3. The process of claim 2, wherein the beds are packed into a set of columns housed in a simulated moving bed apparatus consisting of a feed loading zone, a raffinate elution zone, and a regeneration zone, wherein (a) the feed is loaded onto a first column segment defining the feed loading zone, (b) the 1,5-pentanediamine is eluted from a second column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segments, (c) lysine and the conjugated anion of the salt of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the column is regenerated in the regeneration zone, wherein the raffinate elution zone is not in fluid communication with the feed loading zone, and the regeneration zone is not in fluid communication with the raffinate elution zone.

15. 15. The process of claim 14, wherein a water wash is introduced into the column in a segment upstream of the first column segment defining the feed loading zone, a hydroxide salt solution is introduced into the column in a segment within the raffinate elution zone, and water is introduced into a column segment in the regeneration zone.

16. 15. The process of claim 14, wherein the lysine salt is lysine phosphate.

17. 15. The process of claim 14, wherein the simulated moving bed apparatus comprises 12 column segments, and of the 12 column segments, the feed loading zone comprises 5 column segments, the raffinate elution zone comprises 3 column segments, and the regeneration zone comprises 4 column segments.

18. 18. The process of claim 17, wherein the lysine salt is lysine phosphate.

19. 1. A method for producing 1,5-pentanediamine (PDA), comprising: (a) obtaining a culture broth resulting from fermenting a host microorganism engineered to produce lysine; (b) removing the microorganism from the culture broth; (c) adding a phosphate or sulfate source to the culture broth to form a phosphate or sulfate salt of lysine; (d) contacting the sulfate or phosphate salt-containing culture broth with lysine decarboxylase to decarboxylate the lysine and form a culture broth containing the lysine salt and PDA; (e) contacting the culture broth containing the lysine salt and PDA with a bed of a strong anion exchange resin in a column; (f) eluting the PDA from the column in a first fraction separate from a second fraction containing lysine and lysine salts; wherein the PDA is eluted from the column with a purity of at least 95% and contains less than 5% lysine.

20. 20. The method of claim 19, wherein the column comprises a set of columns housed in a simulated moving bed apparatus consisting of a feed loading zone, a raffinate elution zone, and a regeneration zone, wherein (a) the feed is loaded onto a first column segment defining the feed loading zone, (b) the 1,5-pentanediamine is eluted from a second column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segments, (c) lysine and a conjugate anion of the salt of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the column is regenerated in the regeneration zone, wherein the raffinate elution zone is not in fluid communication with the feed loading zone, and the regeneration zone is not in fluid communication with the raffinate elution zone.

21. 1. A method for forming 1,5-pentanediamine (PDA), comprising contacting a mixture containing at least a 0.3 M solution of a lysine salt with sulfate or phosphate as a conjugate anion with an enzyme having lysine decarboxylase activity for a time sufficient to convert at least 50% of the lysine to PDA.

22. 22. The method of claim 21, wherein the mixture has a pH of 8.0 to 9.

0.

23. 22. The method of claim 21, wherein the mixture contains 0.5 to 2.9 M lysine phosphate at a pH of 8.0 to 9.

0.

24. 24. The method of claim 23, wherein the pH is 8.0 to 8.

5.

25. 22. The method of claim 21, wherein at least 90% of the lysine is converted to PDA.

26. 22. The method of claim 21, wherein the lysine decarboxylase is encoded by the cadA gene of E. coli.

27. 22. The method of claim 21, further comprising separating the PDA from lysine and the conjugated anions by contacting the reaction mixture with a strong base ion exchange resin and eluting the PDA from the resin, wherein the eluted PDA is at least 85% pure, containing less than 15% of the lysine or conjugated anions.

28. 28. The method of claim 27, wherein the strong base ion exchange resin is configured in a simulated moving bed apparatus.

29. 29. The method of claim 28, wherein the simulated migration apparatus comprises a set of columns comprised of a feed loading zone, a raffinate elution zone, and a regeneration zone, wherein (a) the feed is loaded onto a first column segment defining the feed loading zone, (b) the 1,5-pentanediamine is eluted from a second column segment downstream from the feed loading zone relative to the direction of liquid flow through the column segments, (c) lysine and the conjugate anion of the salt of lysine are eluted from a third column segment in the raffinate elution zone, and (d) the column is regenerated in the regeneration zone, wherein the raffinate elution zone is not in fluid communication with the feed loading zone, and the regeneration zone is not in fluid communication with the raffinate elution zone.

30. 30. The process of claim 29, wherein a water wash is introduced into the column in a segment upstream of the first column segment defining the feed loading zone, a hydroxide salt solution is introduced into the column in a segment within the raffinate elution zone, and water is introduced into a column segment in the regeneration zone.

31. 30. The process of claim 29, wherein the lysine salt is lysine phosphate.

32. 30. The process of claim 29, wherein the simulated moving bed apparatus comprises 12 column segments, and of the 12 column segments, the feed loading zone comprises 5 column segments, the raffinate elution zone comprises 3 column segments, and the regeneration zone comprises 4 column segments.

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