WO2012166406A1 - Augmentation du rendement et/ou du débit d'un produit par sursaturation de substrat - Google Patents

Augmentation du rendement et/ou du débit d'un produit par sursaturation de substrat Download PDF

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Publication number
WO2012166406A1
WO2012166406A1 PCT/US2012/038766 US2012038766W WO2012166406A1 WO 2012166406 A1 WO2012166406 A1 WO 2012166406A1 US 2012038766 W US2012038766 W US 2012038766W WO 2012166406 A1 WO2012166406 A1 WO 2012166406A1
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Prior art keywords
tryptophan
mixture
monatin
concentration
retentate
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PCT/US2012/038766
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English (en)
Inventor
Trent H. Pemble
Christopher Solheid
Brent H. Hilbert
AnaLisa DICKEY
Dustin P. JOHNSON
Michael A. Porter
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Cargill, Incorporated
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Publication of WO2012166406A1 publication Critical patent/WO2012166406A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/025—Reverse osmosis; Hyperfiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/02—Reverse osmosis; Hyperfiltration ; Nanofiltration
    • B01D61/027—Nanofiltration

Definitions

  • the present disclosure relates generally to increasing the yield and/or throughput of a product through supersaturation of a solubility-limited substrate. Aspects of the disclosure are particularly directed to increasing the yield and/or throughput of monatin produced in a multi-step equilibrium pathway by increasing the concentration of tryptophan in solution through supersaturation.
  • monatin is also known by a number of alternative chemical names, including: 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid; 4- amino-2-hydroxy-2-(l H-indol-3-y]methy!-pentanedioic acid; 4-hydroxy-4-(3- indoIylmethyI)glutamic acid; and, 3-(l -amino- l ,3-dicarboxy-3-hydroxy-but-4-y!indoie.
  • WO 2003/091396 A2 discloses, inter alia, polypeptides, pathways, and microorganisms for in vivo and in vitro production of monatin.
  • WO 2003/091396 A2 see, e.g., Figures 1 -3 and 1 1 - 13
  • U.S. Patent Publication No. 2005/282260 describe the production of monatin from tryptophan through multi-step pathways involving biological conversions with polypeptides (proteins) or enzymes.
  • high intensity sweeteners allows for the formulation of sweetened beverages with zero or significantly fewer calories, an important health and wellness feature as many countries are attempting to address weight related public health concerns.
  • a naturally occurring high intensity sweetener with a pleasing, sugar-like taste profile, such as monatin is desirable. Since it is desirable to have an economic source of monatin, there is a continuing drive to increase the efficiency of monatin-producing pathways, including the biological multistep pathway described above.
  • the product is monatin and the limited-solubility substrate is tryptophan
  • the multi-step equilibrium pathway includes conversion of tryptophan to indole-3- pyruvate (I3P), I3P to 2-hydroxy 2-(indol-3-yImethyl)-4-keto glutaric acid (MP) and MP to monatin.
  • the yield of monatin is increased by at least about five percent compared to when the tryptophan is at or below its normal solubility limit at the same temperature at the start of the equilibrium pathway.
  • the product is produced by a batch process. In some embodiments, the product is produced by a continuous process.
  • the method above further comprises adding supplemental substrate to the retentate after the concentration of substrate in the retentate falls below a predetermined concentration.
  • the method may further comprise heating a slurry containing the supplemental substrate to a first temperature until the substrate is fully soluble in solution and then cooling the solution to a second temperature that is less than the first temperature.
  • the method may further comprise increasing solubility of the supplemental substrate in solution such that the concentration in solution is higher than its normal solubility limit.
  • the method above further comprises adding supplemental substrate to the retentate to maintain the concentration of substrate in the retentate at a constant concentration.
  • the substrate may be tryptophan and the concentration may be between about 100 and about 1 30 mM.
  • a method of increasing production of monatin in a multi-step equilibrium pathway includes combining water, tryptophan, pyruvate, a first enzyme and a second enzyme to form a mixture, and increasing a solubility of tryptophan in the mixture beyond its normal solubility limit at the same temperature.
  • increasing the solubility of tryptophan beyond its normal solubility limit includes contacting the mixture with a membrane to retain a second mixture having an increased concentration of tryptophan in solution compared to the first mixture.
  • the concentration of tryptophan in the second mixture may be at least three times greater than its normal solubility limit at the same temperature.
  • the concentration of tryptophan in the second mixture may be at least about 1 15mM at about 1 5 degrees Celsius.
  • increasing the solubility of tryptophan beyond its normal solubility limited is performed after the ractions to convert tryptophan to monatin have reached equilibrium. In some embodiments of the method above, increasing the solubility of tryptophan beyond its normal solubility limit is performed at least about 24 hours after the mixture comprising water, tryptophan , pyruvate, a first enzyme and a second enzyme is formed.
  • a method of using a membrane to produce monatin includes combining water, tryptophan, pyruvate, a first enzyme and a second enzyme to form a mixture in which the tryptophan in the mixture is at a first concentration, and contacting the mixture with a membrane, resulting in a retentate comprising tryptophan at a second concentration that is greater than the first concentration.
  • the second concentration may be greater than tryptophan's normal solubility limit at the same temperature. In some embodiments, the second concentration is at least three times greater than the normal solubility limit at the same temperature.
  • the monatin is produced by a continuous process after a given start-up time.
  • the method may further comprise adding supplemental tryptophan to the retentate to maintain a constant concentration of tryptophan in the retentate.
  • the method may further comprise adding supplemental tryptophan to the retentate after the concentration of tryptophan in the retentate falls below a predetermined concentration.
  • the supplemental tryptophan added to the retentate is fully soluble in solution and is at a concentration greater than its normal solubility limit at the same temperature.
  • the method further comprises removing a portion of the retentate.
  • the monatin is produced by a batch process.
  • the method may further comprise leaving the mixture for a time sufficient to reach equilibrium prior to contacting the mixture with a membrane.
  • the mixture may be left for at least about 24 hours; in other embodiments, the mixture may be left for at least about 36 hours.
  • the method above further comprises leaving the mixture for at least about 24 hours after contacting the mixture with the membrane.
  • the method above further comprises contacting the mixture with the membrane a second time.
  • a method of supersaturating tryptophan is used in a batch process for the production of monatin. In some embodiments, a method of supersaturating tryptophan is used in a continuous or semi-continuous process for the production of monatin.
  • monatin is produced in a multi-step equilibrium pathway in which tryptophan is converted to indole-3- pyruvate (I3P), I3P is converted to 2-hydroxy 2-(indoi-3-y!methyl)-4-keto glutaric acid (MP), and MP is converted to monatin.
  • the tryptophan is D-tryptophan and the monatin is a steroisomericaliy-enriched R,R monatin.
  • the first enzyme is an aminotransferase and the second enzyme is an aldolase.
  • the membrane is a reverse osmosis membrane. In some aspects of some or all of the embodiments of the invention, the membrane is a nanofiltration membrane. In some aspect of some or ali of the embodiments of the invention, the mixture contacting the membrane is at a pressure between about 500 and about 900 psi.
  • the retentate after the mixture contacts the membrane, the retentate comprises at least about 90 percent of the tryptophan contained in the mixture; in other aspects, the retentate comprises at least about 95 percent of the tryptophan contained in the mixture. In some aspects of some or all of the embodiments of the invention, after the mixture contacts the membrane, a volume of the retentate is about 10 to about 40 percent of the volume of the mixture; in other aspects, the volume is about 30 to about 35 percent of the volume of the mixture.
  • Monatin has an excellent sweetness quality, and depending on a particular composition, monatin may be several hundred times sweeter than sucrose, and in some cases thousands of times sweeter than sucrose.
  • Monatin has four stereoisomeric configurations which exhibit differing levels of sweetness.
  • the S,S stereoisomer of monatin is about 50-200 times sweeter than sucrose by weight.
  • the R,R stereoisomer of monatin is at least about 2000-2400 times sweeter than sucrose by weight.
  • monatin is used to refer to compositions including any combination of the four stereoisomers of monatin (or any of the salts thereof), including a single isomeric form.
  • solubility-limited in reference to a solubility-limited substrate or molecule means the substrate or molecule becomes insoluble in water at a particular temperature after a given concentration is reached.
  • normal solubility limit means the maximum concentration at which the substrate or molecule is fully soluble in water at a particular temperature, and may also be known as the saturation concentration.
  • fully soluble means all of the substrate or molecule is dissolved in water, and no crystals or appreciable crystalline or precipitate structure are present.
  • the term "supersaturated” or “supersaturation” means the substrate or molecule is fully soluble and metastab!e in water at a concentration that is greater than its normal solubility limit at a particular temperature, it is recognized that when reference is made herein to a 'stable' solution that is supersaturated, the solution is actually in a metastable state,
  • Monatin may be synthesized in whole or in part by one or more of a biosynthetic pathway, chemically synthesized, or isolated from a natural source. If a biosynthetic pathway is used, it may be carried out in vitro or in vivo and may include one or more reactions such as the equilibrium reactions provided below as reactions (I )-(3). In one embodiment is a biosynthetic production of monatin via enzymatic conversions starting from tryptophan and pyruvate and following the three equilibrium reactions below:
  • HMO hydroxymethyl-oxo-glutarate
  • HMG hydroxymethylglutamate
  • reaction ( 1 ) tryptophan and pyruvate are enzymatically converted to indole-3-pyruvate (13 P) and alanine in a reversible reaction.
  • an enzyme here an aminotransferase, is used to facilitate (catalyze) this reaction.
  • tryptophan donates its amino group to pyruvate and becomes BP.
  • reaction (3 ) the amino group acceptor is pyruvate, which then becomes alanine as a result of the action of the aminotransferase.
  • the amino group acceptor for reaction (1 ) is pyruvate; the amino group donor for reaction (3) is alanine.
  • indole-3-pyruvate in reaction (1 ) can also be performed by an enzyme that utilizes other a-keto acids as amino group acceptors, such as oxaloacetic acid and a-keto-glutaric acid.
  • the formation of monatin from MP (reaction (3)) can be performed by an enzyme that utilizes amino acids other than alanine as the amino group donor. These include, but are not limited to, aspartic acid, glutamic acid, and tryptophan.
  • reaction (3) Some of the enzymes useful in connection with reaction (1 ) may also be useful in connection with reaction (3).
  • aminotransferase may be useful for both reactions (1 ) and (3).
  • the equilibrium for reaction (2), the aldolase-mediated reaction of indole-3-pyruvate to form MP i.e. the aldolase reaction
  • reaction (3) The equilibrium constants of the aminotransferase-mediated reactions of tryptophan to form indole-3-pyruvate (reaction ( 1 )) and of MP to form monatin (reaction (3)) are each thought to be approximately one. Methods may be used to drive reaction (3) from left to right and prevent or minimize the reverse reaction. For example, an increased concentration of alanine in the reaction mixture may help drive forward reaction (3).
  • a multi-step pathway refers to a series of reactions that are linked to each other such that subsequent reactions utilize at least one product of an earlier reaction.
  • the substrate (for example, tryptophan) of the first reaction is converted into one or more products, and at least one of those products (for example, indoIe-3-pyruvate) can be utilized as a substrate for the second reaction.
  • the three reactions above are equilibrium reactions such that the reactions are reversible.
  • a multi-step equilibrium pathway is a multi-step pathway in which at least one of the reactions in the pathway is an equilibrium or reversible reaction.
  • R.R stereoisomer of monatin is the sweetest of the four stereoisomers, it may be preferable to selectively produce R,R monatin.
  • the focus is on the production of R.R monatin.
  • the present disclosure is applicable to the production of any of the stereoisomeric forms of monatin (R,R; S,S; S,R; and R,S), alone or in combination.
  • the monatin consists essentially of one stereoisomer - for example, consists essentially of S,S monatin or consists essentially of R,R monatin.
  • the monatin is predominately one stereoisomer - for example, predominately S,S monatin or predominately R,R monatin.
  • "Predominantly” means that of the monatin stereoisomers present in the monatin, the monatin contains greater than 90% of a particular stereoisomer.
  • the monatin is substantially free of one stereoisomer - for example, substantially free of S,S monatin.
  • “Substantially free” means that of the monatin stereoisomers present in the monatin, the monatin contains less than 2% of a particular stereoisomer.
  • the monatin is a stereoisomericaily-enriched monatin mixture.
  • “Stereoisomericaily-enriched monatin mixture” means that the monatin may contain more than one stereoisomer and at least 60% of the monatin stereoisomers in the mixture is a particular stereoisomer. In other embodiments, the monatin contains greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer.
  • a monatin composition comprises a stereoisomericaily-enriched R,R-monatin, which means that the monatin comprises at least 60% R,R monatin.
  • stereoisomericaily-enriched R.R-monatin comprises greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R,R monatin.
  • the starting material may be D-tryptophan
  • the enzymes may be a D- aminotranferase and an R-specific aldolase.
  • the three reactions, which are shown be!ow, may be carried out in a single reactor or a multiple-reactor system.
  • the two enzymes i.e. the D- aminotransferase and the R-specific aldolase
  • the two enzymes may be added at the same time and the three reactions may run simultaneously.
  • the same enzyme may be used to catalyze reactions (6) and (8).
  • a D-aminotransferase is an enzyme with aminotransferase activity that selectively produces, in the reactions shown above, D-alanine and R,R-monatin.
  • An R-specific aldolase is an enzyme with aldolase activity that selectively produces R-MP, as shown in reaction (7) above.
  • L-alanine may react with the L-aminotransferase to produce R,S-monatin
  • D-alanine may react with 13 to form D-tryptophan, resulting in a racemate of L-tryptophan and D-tryptophan, which has poor solubility.
  • the method and system described herein for increasing monatin yield and/or throughput by supersaturation of tryptophan is applicable to monatin produced using alternative pathways to what is disclosed herein.
  • the focus herein is on the production of R,R monatin starting from D-tryptophan, however, it is recognized that the method and systems described herein are applicable to starting with L-tryptophan.
  • the resulting monatin produced using the method described above may be present in a mixture that contains other components, including starting materials, intermediates, side products of the monatin-producing reactions or combinations thereof. It is preferable to separate the monatin from these other components, which may include, for example, tryptophan, pyruvate, alanine, I3P, MP, HMG and HMO. It may be preferable to separate and purify monatin from this mixture in order to achieve a particular purity of monatin; such separation and purification methods are not the focus of the present disclosure.
  • monatin may be produced by the three step pathway disclosed above, and each of the three reactions are reversible reactions. As such, it may be challenging to produce a high yield of monatin, particularly given that some of the intermediates may be unstable and side reactions may also occur. Moreover, the solubility of tryptophan, one of the starting materials or substrates in the disclosed pathway, is a limiting factor in the conversion of tryptophan to monatin. Given the significant commercial need to maximize monatin yield, a method of increasing the solubility of tryptophan is desirable. The present disclosure focuses on a method and system for increasing the concentration of tryptophan available for the monatin-producing reactions through supersaturation. As shown below, supersaturation of tryptophan results in an increase in monatin yield. As described below, tryptophan supersaturation may be achieved by various methods and at different points in the process of producing monatin.
  • the monatin-producing reactions described above are commonly performed at a temperature between about 1 0 degrees Celsius and about 25 degrees Celsius. In some embodiments, the reactions are performed at a temperature of about 15 degrees Celsius.
  • the solubility of tryptophan varies, in part, as a function of the temperature of the tryptophan/solvent mixture.
  • the normal solubility limit of L-tryptophan or D-tryptophan (separately) in water is approximately 35 mM ⁇ 5 mM at 15 °C.
  • a mixture of L-tryptophan and D-tryptophan has a different solubility limit.
  • solubility of tryptophan when reference is made to the solubility of tryptophan, it is recognized that reference is made to either the L- enantiomer or the D- enantiomer of tryptophan, but not to a mixture of the enantiomers. It is recognized that the normal solubility limit of tryptophan at a given temperature will also depend on the presence of salts and other ions and components in the solvent mixture.
  • Methods may be used to supersaturate tryptophan or increase the solubility of tryptophan above its normal solubility limit.
  • Supersaturation is a metastable state and may be used to increase monatin yield and/or throughput.
  • a higher concentration of tryptophan in solution means there is more tryptophan available to be converted to indole-3-pyruvate (13P) and ultimately monatin. In other words, having more tryptophan in solution shifts the three equilibrium reactions in the forward direction, resulting in a higher production of monatin.
  • Supersaturation as exhibited below, allows for a significantly higher concentration of tryptophan available in the solution to react with the enzymes and perform the conversions outlined above.
  • the maximum stable concentration of tryptophan in a solution may be more or less depending on the presence of other salts in the solution.
  • tryptophan may be supersaturated thermally by increasing the temperature of a solution containing tryptophan.
  • supersaturation may be achieved by removing water from a solution containing tryptophan (i.e. dewatering), for example, by using a membrane.
  • tryptophan supersaturation is achieved by increasing the temperature of a slurry of tryptophan (a combination of soluble and insoluble tryptophan) in water until essentially all of the tryptophan is dissolved in solution. The solution is then cooled to a desired operating temperature for performing the monatin-producing reactions (for example, about 1 5 degrees Celsius).
  • a desired operating temperature for performing the monatin-producing reactions for example, about 1 5 degrees Celsius.
  • the soluble concentration of tryptophan achievable through supersaturation will depend in part on the temperature that the slurry is heated to. It is recognized that increasing the temperature to a temperature greater than the operating temperature will result in increased solubility of tryptophan. Thus an increased concentration of soluble tryptophan results from heating the slurry above about 20 degrees Celsius.
  • the tryptophan slurry may be heated to a temperature ranging between about 20 and about 120 degrees Celsius; in other embodiments, between about 80 and about 105 degrees Celsius. In some embodiments, the slurry is heated to about 100 degrees Celsius. As demonstrated in the Examples below, a stable solution of tryptophan at a concentration of 130 mM was achieved in water at 1 5 degrees Celsius, after the slurry was heated to about 100 degrees Celsius. As stated above, the normal solubility limit of tryptophan in water at 15 degrees Celsius is about 35 mM. Thus soluble concentrations of tryptophan ranging between 40 and 130 mM can be achieved through thermal supersaturation.
  • the tryptophan slurry may also include pyruvate and the enzymes for the monatin-producing reactions. Supersaturation of tryptophan is achieved as described above - heating at a given temperature and then cooling. The temperature may be limited due to the thermal stability of the enzymes, thus the slurry may be heated to a temperature in which the enzymes are still thermally stable.
  • tryptophan supersaturation may be achieved thermally by starting with a dilute solution of tryptophan (rather than a slurry).
  • the dilute solution is heated at a temperature that causes evaporation of excess water in the solution until a target concentration of tryptophan is reached.
  • a membrane is used to remove water from a mixture comprising at least tryptophan and water.
  • the membrane is selected based on its ability to retain tryptophan, thus creating a retentate having a concentration of tryptophan that is higher than the concentration of tryptophan in the original mixture.
  • the membrane may be selected based on its molecular weight cut-off. In some embodiments, over 90% of the tryptophan is retained by the membrane; in other embodiments, over 95% of the tryptophan is retained by the membrane.
  • the mixture passing over the membrane not only comprises tryptophan and water, but also comprises other substrates, intermediates and enzymes found in the monatin-producing reactions.
  • the membrane is selected based on its ability to retain all of the carbon-containing molecules, while letting water pass through the membrane. In some embodiments, over 90% of the carbon-containing molecules are retained by the membrane; in other embodiments, over 95% of the carbon-containing molecules are retained by the membrane.
  • the membrane is a reverse osmosis (RO) membrane. More specifically, in one embodiment, the RO membrane is a tubular RO membrane, such as, for example, AFC 99 from PCI Membranes. In another embodiment, the RO membrane is a spiral wound RO membrane, such as, for example, SW30-4040 from DOW Filmtec. In some embodiments, the membrane is a nanofiltration membrane, such as, for example, DL I 812 from GE.
  • RO reverse osmosis
  • the particular membrane selected depends, in part, on the composition of the mixture, the target composition of the retentate, and the volume and flow rate of the mixture.
  • a method of using a membrane includes creating a mixture that includes water, tryptophan and pyruvate, as well as the enzymes that facilitate the reactions in the multi-step equilibrium pathway. After creating the mixture, a sufficient amount of time passes such that equilibrium of the reactions is reached and the mixture is fully soluble. At that point, the mixture includes monatin, tryptophan, pyruvate, alanine, I3P, MP, HMO and HMG.
  • the time to reach equilibrium is at least 24 hours; in other embodiments, the time to reach equilibrium is at least 36 hours; and in yet other embodiments, at least 48 hours.
  • the mixture is passed across or comes into contact with a membrane.
  • the water permeates through the membrane while the tryptophan and other carbon-containing molecules, including monatin, are retained by the membrane (forming a retentate).
  • the tryptophan and other carbon-containing molecules, including monatin are retained by the membrane (forming a retentate).
  • the membrane dewatering step concentrates the components in the retentate, thereby perturbing the previously reached equilibrium, resulting in a new equilibrium.
  • the monatin yield of the mixture was 1 1 ,7%.
  • the monatin yield of the retentate was 16.7%, resulting in a 5% yield increase.
  • the retentate was then held for about five days and the monatin yield was 20.7%, resulting in an additional 4% increase.
  • the mass increase of monatin as a result of the dewatering step and the five-day hold was 77%.
  • the hold step after the RO concentration or dewatering step allows time for the enzymes to perform the monatin-producing reactions.
  • the monatin-producing reactions are occurring. It is recognized that the monatin yield increase will depend in part on how long it takes to complete the dewatering step and how long the retentate is held after completion of the dewatering step.
  • the monatin yield of a mixture including monatin, tryptophan, pyruvate, alanine, 13 P, MP, HMO and HMG measured about 36 hours after being created was 16.4%.
  • the monatin yield of the retentate was 20.1 %.
  • the mass increase of monatin as a result of the deatering step was 22%.
  • the volume of the retentate is equal to about 30 to about 35 percent of the volume of the original mixture.
  • the concentration of the components in the retentate, including tryptophan, immediately following the dewatering step is at least 3 times higher than the concentration in the mixture.
  • the retentate may be further concentrated, depending in part, on the composition of the original mixture. The extent of concentration also depends, in part, on the desired concentration of tryptophan in the retentate.
  • the volume of the retentate is between about 1 and about 20 percent of the volume of the original mixture; in other embodiments, between about 10 and about 40 percent of the volume of the original mixture.
  • the membrane dewatering step is performed before the mixture, containing water, tryptophan, pyruvate, and the monatin-producing enzymes, has reached equilibrium.
  • the membrane system is activated and continues until a desired concentration of tryptophan (for example, between about 1 15 m and about 130 mM) is reached.
  • the membrane system may then be shut off and in some embodiments, the retentate is left or held for a period of time, for example, 24 hours, during which the monatin- producing reactions are occurring.
  • a second dewatering step may be performed to concentrate the tryptophan back up to the desired concentration. Even with a second dewatering step, in some cases, this method may be completed in a shorter period of time as compared to the methodology in which dewatering is not performed until after the mixture has reached equilibrium. A favorable monatin yield is observed using this method.
  • additional dewatering steps may be performed. The number of dewatering steps and the hold time (if any) may be determined based in part on the desired monatin yield and optimizing the overall efficiency of the process for producing monatin.
  • a driving force of the membrane dewatering step is the pressure difference between the feed side of the membrane and the permeate side of the membrane.
  • the inlet pressure of the mixture being fed into the membrane will depend, in part, on the feed flow rate, the type and size of the membrane, as well as the composition of the mixture. In some embodiments, the inlet pressure is between about 200 and about 1500 psi; in other embodiments, the inlet pressure is between about 500 and about 900 psi. In some embodiments, the mixture may contain a higher salt content, which may impact the operating pressure.
  • a method of supersaturating tryptophan includes starting with a tryptophan-containing slurry, using heat to create a soluble system and then further increasing a concentration of tryptophan using a membrane.
  • additional tryptophan or supplemental tryptophan is added to the mixture (containing water, tryptophan, pyruvate, intermediates, monatin and enzymes) as the tryptophan is consumed in the monatin-producing reactions and the tryptophan concentration decreases.
  • Supplemental tryptophan may be added in order to maintain or return the tryptophan to a supersaturated level. This results in an increased monatin yield and/or throughput since maintaining or returning the tryptophan to a higher concentration continues to drive the monatin- producing reactions forward.
  • the supplemental tryptophan may be at a supersaturated concentration prior to being added to the mixture, and the supersaturation may be achieved thermally, by membrane dewatering, or a combination of the two. In some embodiments, all or a portion of the supplemental tryptophan may be recycled from downstream in the process (for example, following separation and/or purification steps not focused on herein). In some embodiments, the supplemental tryptophan may be continuously fed to the mixture to maintain tryptophan at a particular concentration. For example, it may be desired to maintain tryptophan at a concentration ranging between about 100 and about 130 mM. In some embodiments, supplemental tryptophan may be periodically added to the mixture, for example, when the concentration in the mixture falls be!ow a predetermined concentration level.
  • the method includes continuously operating a membrane system combined with a feed/retentate tank containing the mixture with water, tryptophan, pyruvate, intermediates, monatin and enzymes. Because the membrane system is continuously running, the mixture is also the retentate. Once the enzymes are added to the feed tank, the monatin-producing reactions are initiated and thus the retentate also includes monatin. Water is removed from the system as permeate, resulting in an increased or supersaturated concentration of tryptophan.
  • an additional volume of the retentate/reaction mixture may be continuously removed from the retentate side of the membrane.
  • the removed-retentate may then undergo additional processing to separate and/or purify monatin from the additional components in the removed- retentate.
  • supplemental tryptophan is added back to the feed tank after a given start-up time.
  • pyruvate and enzymes are also added to the feed tank.
  • the supplemental tryptophan may be a dilute solution (at or below the norma! solubility limit) or a supersaturated solution, which may be achieved thermally, by membrane dewatering, or a combination thereof.
  • the supplemental tryptophan may be added in order to maintain tryptophan at a predetermined concentration in the retentate, for example, between about 100 and about 130mM. All or a portion of the supplemental tryptophan may be recycled from downstream in the process. All or a portion of the pyruvate and enzymes added back to the feed tank may also be recycled from downstream in the process.
  • the presence of salts and other ions in solution will impact the concentration of soluble tryptophan achievable through supersaturation. It is also recognized that other components, such as cofactors, substrates, etc., which may be present in a tryptophan- containing mixture described herein may also impact the supersaturation concentration of tryptophan.
  • the salts may create nucleation sites that initiate or propagate the formation of tryptophan crystals.
  • the monatin produced was predominantly R,R monatin.
  • a series of suspensions of D-tryptophan (>99% purity) in water from 6.12 g/L to 40.8 g/L (nominally 30 to 200 mM) in increments of 2.04 g/L (nominally 10 mM) were prepared in 20 mL tubes with threaded caps and heated to 100°C to fully dissolve the D-tryptophan. Weights and volumes of D-tryptophan and water are given in Table 1. After heating, the solutions were then placed in a water bath at about 15°C for about 72 hours.
  • the bioreaction mixture was transferred from the bioreaction vessel to a nitrogen blanketed tank. Prior to performing the dewatering or concentration step, the composition of the bioreaction mixture was sampled and the results are shown in Table 4. The total volume of the mixture in Table 4 remained at about 10 L. Tabie 4. Composition of Bioreaetion Mixture after 40 hours
  • the mixture was then concentrated on a tubular RO membrane having 0.024 m 2 surface area, AFC99 RO-type from PCI Membranes.
  • the system consisted of the feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at about 500 psi inlet pressure, and the feed tank was under a constant nitrogen blanket. The target operating temperature was 15°C, however, the temperature was not monitored.
  • the monatin yield of the mixture in Table 5 was 16.7%.
  • the dewatering or concentration step increased the monatin yield by 5%.
  • the monatin yield of the mixture in Table 6 was 20.7%. Thus an additional yield increase of 4% was observed by leaving the mixture for a significant time period following the dewatering step. A yield increase of 9% was observed through the combination of dewatering followed by a hold. The monatin mass after the hold, as compared to the monatin mass after 40 hours (see Table 4), resulted in a monatin mass increase of 77% ((93.90 g - 53,07 g)/53.07 g).
  • the D-tryptophan and pyruvate fully went into solution.
  • the concentration of D- tryptophan in the solution was 28.4 inM, which is below the normal solubility limit.
  • the test solution was then concentrated on a tubular AFC99 RO membrane, having 0.88 m 2 surface area, from PCI Membranes.
  • the system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and two permeate collection vessels.
  • the system was operated at 500 psi inlet pressure and an average temperature of about 18.5°C, and the feed tank was under a constant nitrogen blanket. After the concentration was completed, nitrogen was used to displace the solution remaining in the membrane housing back into the feed (retentate) tank.
  • a membrane flush of 10 L was completed to recover the remaining tryptophan and pyruvate to determine how much stayed behind in the housing and on the membrane surface.
  • the total volume of the retentate was about 24.0 L.
  • the composition of the retentate is shown in Table 8. D-tryptophan was supersaturated to a concentration of 1 15.5 mM No tryptophan or pyruvate was measured in the permeate while the membrane flush sample had about 19.0 g of tryptophan and about 14.8 g of pyruvate.
  • the mixture was next concentrated on a tubular AFC99 RO membrane, having 0.88 m 2 surface area, from PCI Membranes.
  • the system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at 500 psi inlet pressure and the feed tank was under a constant nitrogen blanket.
  • the monatin yield of the mixture in Table 12 was 18.8%.
  • giving the enzymes additional time to facilitate the monatin-producing reactions after the tryptophan was supersaturated resulted in an additional 2, 1 % yield increase.
  • a yield increase of 5.2% was observed through the combination of a second dewatering step followed by a hold.
  • the mixture was then concentrated on a tubular RO membrane, having 0.88 m 2 surface area, AFC99 RO-type from PCI Membranes.
  • the system consisted of a feed tank with a cooling coil, a feed pump with inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at about 500 psi inlet pressure, and the feed tank was under a nitrogen blanket. The target operating temperature was about 15°C S however, over time the temperature increased to about 27°C.
  • Table 15 shows the composition of the retentate after the dewatering or concentration step.
  • the total volume of the retentate was about 7.8 L.
  • the mixture from Table 15 was evaluated about 48 hours after the enzymes were first added to the bioreaction vessel.
  • the monatin yield of the mixture in Table 1 was 20.1 %, resulting in a 3.7% yield increase.
  • the monatin yield of the mixture in Table 1 8 was 1 7.5%, resulting in a 1 .1 % yield increase.
  • the concentration of the solution i.e. dewatering
  • the test solution was concentrated on a spiral wound SW30 4040 RO membrane, having a surface area of 7.4 m 2 , from DOW Filmtec.
  • the concentration system consisted of a feed tank with a cooling coil, a feed pump with an inline heat exchanger, a membrane housing, and a permeate collection tank. The system was operated at 700 psi inlet pressure and about 33°C, and the feed tank was under a nitrogen blanket.
  • the mixture/retentate from Table 20 was then concentrated a second time using the same RO membrane system from the initial concentration. The temperature was about 16.5 °C during this second concentration. The mixture/retentate was concentrated until the concentration of tryptophan in the new retentate reached 1 15 mM, The membrane system was shut off and a sample was taken. The composition of the sample is shown in Table 21 . The volume of the mixture was 10.9 L.
  • the mixture/retentate from Table 24 was then concentrated a second time using the same RO membrane system from the initial concentration. The temperature was about 14°C during this second concentration. The mixture/retentate was concentrated until the concentration of D- tryptophan in the new retentate reached 1 15 mM The membrane system was shut off and a sample was taken. The composition of the sample is shown in Table 25. The volume of the mixture was
  • aldolase active enzyme supplied as 75 grams of MONO 18 cell free extract
  • the solution was then concentrated using a DL1812 nanofiltration (NF) membrane from GE.
  • NF nanofiltration
  • the system was operated at an inlet pressure of about 500 psi and about 15 °C.
  • the membrane system was operated until the volume of the retentate was about 2.6 L, which was about 29 minutes.
  • the concentration of tryptophan in the retentate was 93.4 mM and the mass was 50.2 g.
  • the solution was stable at this concentration for over four days at about 15 °C.
  • the mass balance is 82% for the permeate and the retentate.
  • the membrane did allow some of the D-tryptophan to pass through the membrane which explains why the mass of D- tryptophan in the retentate was lower than the mass in the initial solution. It is assumed that the missing D-tryptophan was left on the surface of the membrane which could be recovered with a rinse step.

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Abstract

L'invention concerne des procédés et des systèmes pour augmenter le rendement et/ou le débit d'un produit obtenu dans une voie à l'équilibre multi-étapes. Le rendement et/ou le débit du produit est accru par sursaturation d'un substrat à solubilité limitée. Une sursaturation peut être obtenue, dans certains modes de réalisation, grâce à une déshydratation de membrane. Dans certains modes de réalisation, le produit est la monatine et le substrat est le tryptophane.
PCT/US2012/038766 2011-05-31 2012-05-21 Augmentation du rendement et/ou du débit d'un produit par sursaturation de substrat WO2012166406A1 (fr)

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US10030177B2 (en) 2011-05-27 2018-07-24 Cargill, Incorporated Bio-based binder systems
US10144902B2 (en) 2010-05-21 2018-12-04 Cargill, Incorporated Blown and stripped blend of soybean oil and corn stillage oil

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US5403604A (en) * 1991-10-15 1995-04-04 The Nutrasweet Company Sugar separation from juices and product thereof
JP2000086600A (ja) * 1998-09-04 2000-03-28 Dainichiseika Color & Chem Mfg Co Ltd 高濃度アミノ酸溶液の調製方法および高濃度アミノ酸溶液を用いたペプチドの合成方法
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JP2009538145A (ja) * 2006-05-24 2009-11-05 カーギル・インコーポレイテッド 平衡反応の収量を増加させるための方法およびシステム
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US4837371A (en) * 1986-09-08 1989-06-06 Mitsui Toatsu Chemicals, Inc. Process for concentration of an aqueous solution of amino acid
US5403604A (en) * 1991-10-15 1995-04-04 The Nutrasweet Company Sugar separation from juices and product thereof
US20030059901A1 (en) * 1997-11-26 2003-03-27 Novozymes A/S Process for isomaltose production
JP2000086600A (ja) * 1998-09-04 2000-03-28 Dainichiseika Color & Chem Mfg Co Ltd 高濃度アミノ酸溶液の調製方法および高濃度アミノ酸溶液を用いたペプチドの合成方法
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US20080292775A1 (en) * 2007-05-22 2008-11-27 The Coca-Cola Company Delivery Systems for Natural High-Potency Sweetener Compositions, Methods for Their Formulation, and Uses

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US10144902B2 (en) 2010-05-21 2018-12-04 Cargill, Incorporated Blown and stripped blend of soybean oil and corn stillage oil
US10851326B2 (en) 2010-05-21 2020-12-01 Cargill, Incorporated Blown and stripped blend of soybean oil and corn stillage oil
US11339347B2 (en) 2010-05-21 2022-05-24 Cargill, Incorporated Blown and stripped blend of soybean oil and corn stillage oil
US11884894B2 (en) 2010-05-21 2024-01-30 Cargill, Incorporated Blown and stripped blend of soybean oil and corn stillage oil
US10030177B2 (en) 2011-05-27 2018-07-24 Cargill, Incorporated Bio-based binder systems
US10550294B2 (en) 2011-05-27 2020-02-04 Cargill, Incorporated Bio-based binder systems
US11814549B2 (en) 2011-05-27 2023-11-14 Cargill, Incorporated Bio-based binder systems

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