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EP1032699A1 - Acide gamma-polyglutamique (gamma-pga) a masse moleculaire elevee - Google Patents

Acide gamma-polyglutamique (gamma-pga) a masse moleculaire elevee

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Publication number
EP1032699A1
EP1032699A1 EP98960252A EP98960252A EP1032699A1 EP 1032699 A1 EP1032699 A1 EP 1032699A1 EP 98960252 A EP98960252 A EP 98960252A EP 98960252 A EP98960252 A EP 98960252A EP 1032699 A1 EP1032699 A1 EP 1032699A1
Authority
EP
European Patent Office
Prior art keywords
pga
molecular weight
solution
cells
cell culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98960252A
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German (de)
English (en)
Inventor
Alex Diiorio
Louis Stock
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Worcester Polytechnic Institute
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Worcester Polytechnic Institute
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Publication of EP1032699A1 publication Critical patent/EP1032699A1/fr
Withdrawn legal-status Critical Current

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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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • ⁇ -PGA ⁇ -Poly (glutamic acid), hereinafter " ⁇ -PGA”
  • ⁇ -PGA can be synthesized as a capsule by certain microorganisms, such as Bacillus licheniformis (Bacillus subtilis) using specific media formulations.
  • the utility and yield of the polymer have been limited by, among other factors, the limited and heterogenous size of the polymer compounded by uncontrolled degradation of ⁇ -PGA that typically occurs consequent to isolation and purification steps.
  • ⁇ -PGA having a molecular weight no greater than 2.5xl0 6 g/mol generally is separated from cell cultures by processing the cells in a high-shear apparatus, such as a blender.
  • the invention is directed to a method of synthesizing ⁇ -PGA of a high molecular weight, for separating ⁇ -PGA from cells of a cell culture and to a method for fractionating ⁇ -PGA from a ⁇ -PGA-containing cell culture.
  • the invention also is directed to ⁇ -PGA of high molecular weight and a purified ⁇ -PGA composition.
  • high molecular weight ⁇ -PGA is produced using a broth comprising trisodium citrate.
  • high molecular weight ⁇ -PGA is produced using a starting inoculum of cells cryopreserved from a culture grown in a medium that includes trisodium citrate.
  • the method of producing high molecular weight ⁇ -PGA comprises culturing Bacillus licheniformis cells in a broth comprising trisodium citrate, cryopreserving the cultured cells as a 1:1 colution with 20% glycerol and culturing the cryopreserved cells, whereby the cells produce high molecular weight ⁇ -PGA.
  • the present invention is further directed toward cryopreserved Bacillus licheniformis bacteria, wherein the bacteria have been cultured in broth comprising trisodium citrate and wherein the cultured bacteria have been preserved in a 1:1 solution with 20% glycerol.
  • high molecular weight ⁇ -PGA is produced using a broth comprising citric acid monohydrate, wherein the citric acid monohydrate has been dissolved in water and the pH of the citric acid solution adjusted to about pH 6.0 prior to its addition to the broth.
  • the present invention is also drawn to a method of separating ⁇ -PGA from the cells of a ⁇ -PGA-producing cell culture.
  • the method of separating ⁇ -PGA from cells of a cell culture includes lowering the pH of the cell culture to about pH 2. The ⁇ -PGA is then separated from the cells.
  • Another embodiment of the invention includes fractioning the ⁇ -PGA directly from a cell culture by lowering the pH of the cell culture to about pH 2 and subjecting the cell culture to tangential flow filtration.
  • the invention relates to a method for increasing viscosity of a ⁇ -PGA solution. The method includes treating the ⁇ -PGA solution with a chelator to remove metal ions bound to the ⁇ -PGA and then removing the chelator, resulting in ⁇ -PGA having higher viscosity and lower metal ion content.
  • the present invention is directed to purified ⁇ -PGA having a molecular weight of at least about 2.5 x 10 6 g/mol.
  • the present invention provides purified ⁇ -PGA composition having a relatively high molecular weight.
  • High molecular weight ⁇ -PGA is produced using the production media described herein or the cryopreserved cells as described herein.
  • Lowering of the pH of the ⁇ -PGA- containing cell culture allows separation of the high molecular weight ⁇ -PGA from the cells under relatively low- shear conditions, such as centrifugation at low g-force, or filtration using a low shear filtration device resulting in higher average molecular weight of the isolated ⁇ -PGA, less contamination with bacterial cell components and better yield.
  • ⁇ -PGA of relatively high viscosity and low metal ion content, thereby potentially providing greater utility for the purified ⁇ -PGA.
  • ⁇ -PGA produced by the method of the invention and having a reduced metal ion content can have a significantly increased capacity to bind metal ions and can be used as a chelator of metal ions.
  • ⁇ -Poly (glutamic acid) includes repeating units of glutamic acid that are linked between ⁇ -amino and ⁇ - carboxylic acid functional groups of the glutamic acid.
  • ⁇ -PGA has the following structural formula:
  • n generally is in the range of between about one thousand and over twenty thousand.
  • a salt of the ⁇ -PGA has the following structural formula:
  • n generally is in the range of between about one thousand and over twenty thousand and "M” can be, for example, a suitable metal, such as sodium.
  • the invention is directed to a method of producing ⁇ -PGA having high molecular weight (for example, a molecular weight greater than 2.5 x 10 6 g/mol) .
  • the invention is directed to preventing significant degradation of ⁇ -PGA molecular weight during isolation and purification.
  • Various embodiments of the method include separation of ⁇ -PGA from a culture broth, and treatment of the ⁇ -PGA after isolation.
  • ⁇ -PGA can be separated from cells, such as Bacillus subtilis or Bacillus licheniformis , cultured in a suitable production broth.
  • a suitable production broth can be formed from a seed broth, such as that described by Kunioka and Gotoh (Applied Microbiology and Biotechnology, 40:1031- 1035 (1993)), and by R.L. Stock, II (Masters Thesis, "Rheological Characterization of (Poly- ⁇ ) Glutamic Acid Fermentations," August 1996, Worcester Polytechnic Institute) .
  • the seed broth is inoculated with B . licheniformis, strain ATCC 9945A, obtained from American Type Tissue Collection. A 2% to 6% dilution of inoculum in seed broth typically is employed.
  • higher viscosities are achieved as a result of employing a higher (6%) concentration of inoculum in the seed broth, indicating higher molecular weight and/or higher yield of polymer.
  • the inoculated seed broth is cultured for a suitable period of time under suitable conditions, until the cells reach a late exponential/early stationary phase.
  • the seed broth then is employed to inoculate a suitable production broth, such as that described in Example II.
  • a 2% to 6% dilution seed culture to production broth typically is employed.
  • a higher proportion of seed culture to production broth is employed to decrease the lag phase to production.
  • the seeded production broth referred to herein as the cell culture
  • the seeded production broth is cultured for a suitable period of time under suitable conditions to allow maximum production of ⁇ - PGA.
  • the culture is incubated for sixty-six hours at 37°C at 250 RPM in a rotary shaker.
  • the cell culture can be fermented in a fermenter, allowing shorter incubation times.
  • the culture is incubated for forty-eight hours at 37 °C.
  • Fermentation time can be reduced by, for example, feeding pure oxygen to the cell culture at no more than about 5 % saturation of the solution with dissolved oxygen.
  • Cell culture viscosity is monitored during incubation and increases during production of ⁇ -PGA. Generally, the viscosity will begin to decrease late in the production phase.
  • the ⁇ -PGA is harvested from the cell culture prior to this decrease to preserve molecular weight and yield.
  • high molecular weight ⁇ -PGA is produced using standard production medium (described in Example I) comprising trisodium citrate at a suitable concentration.
  • the trisodium citrate is present at a concentration of about 50 to about 100 g/L.
  • the trisodium citrate is present at a concentration of about 60 to about 65 g/L.
  • the trisodium citrate is present at a concentration of 61.4 g/L.
  • the high molecular weight ⁇ -PGA is produced using standard production medium wherein the citric acid monohydrate is dissolved in water and the pH of the citric acid solution adjusted to 6.0 with NaOH, prior to its addition to the production medium.
  • high molecular weight ⁇ -PGA is produced using standard production medium wherein the medium is pH adjusted with semi-conductor grade (99.99% pure) NaOH. While not being bound by theory, it is believed that the trisodium salt of citric acid is not as strongly an acidic compound because hydrogen has been replaced by sodium. When using trisodium citrate in culture medium, less sodium hydroxide is used for the final pH adjustments of the medium to 7.0 (normally, because citric acid glutamic acids are present in the medium, large amounts of sodium hydroxide are used for pH adjustment) . Reagent grade sodium hydroxide contains trace metal (and possibly other) contaminants. Therefore, effectively less trace metals or other contaminants may be present in the trisodium medium compared to the standard production medium.
  • a standard production medium formulation protocol calls for addition of all the dry ingredients plus glycerol, followed by pH adjustment to 7.0 with sodium hydroxide.
  • the citric acid added in this form has already been neutralized. Since citric acid has three pKa's or three effective buffering regions, the final form of citric acid present in the medium may be altered when using the trisodium salt of citric acid compared to the monohydrate.
  • cells having been grown in standard production medium comprising trisodium citrate are cryopreserved in about a 1:1 ratio with about 20% glycerol.
  • Cells thus cryopreserved can be grown in standard production medium to obtain high molecular weight ⁇ -PGA.
  • High molecular weight ⁇ -PGA is separated from cells of the cell culture by the method of the present invention.
  • the temperature of the cell culture is reduced to at least room temperature and, thereafter, the pH of the cell culture is adjusted or reduced, by adding a suitable acid, such as concentrated HCl, to about pH 2 or below.
  • the cell culture is then centrifuged under relatively low-shear conditions, such as 10,000 x g or less, to pelletize the bacteria.
  • the cell culture can be diluted with a suitable solvent, such as water, or a solution of citric acid having a concentration of about 0.1 M to about 1 M and at about pH 2, thereby lowering the viscosity and allowing centrifugation at a lower g-force, such as about 6,000 x g.
  • a suitable solvent such as water
  • a solution of citric acid having a concentration of about 0.1 M to about 1 M and at about pH 2, thereby lowering the viscosity and allowing centrifugation at a lower g-force, such as about 6,000 x g.
  • a suitable solvent such as water
  • a solution of citric acid having a concentration of about 0.1 M to about 1 M and at about pH 2
  • a solution of citric acid having a concentration of about 0.1 M to about 1 M and at about pH 2
  • the supernatant is separated from the pelletized material resulting in ⁇ -PGA supernatant.
  • the ⁇ -PGA supernatant can be analyzed for
  • the ⁇ -PGA can be separated from the cells of the cell culture by tangential flow filtration (TFF) .
  • TFF tangential flow filtration
  • a TFF device equipped to handle high viscosity fluids is used (such as a Millipore Prostak ® system) to filter the fermentation broth (relative shear rates are at least 10 times higher in the Millipore Pellicon ® system) .
  • the culture is harvested as described above. This step as opposed to the centrifugation step alone removes all cellular debris, which is critical for pharmaceutical applications.
  • the pH of the resulting ⁇ -PGA supernatant or ⁇ -PGA filtrate can be raised to at least about pH 6 to reduce or minimize hydrolysis of ⁇ -PGA in subsequent processing steps.
  • a suitable base such as NaOH
  • the method of the present invention involves fractionating ⁇ -PGA directly from a cell culture comprising subjecting the cell culture to TFF.
  • the pH of the cell culture is adjusted or lowered with a suitable acid, such as HCl, to about pH 3 or below.
  • the pH-adjusted cell culture is subjected to TFF to remove the bacteria using a Millipore filtration system equipped for high viscosity fluids such as Prostak ® .
  • a fraction of ⁇ -PGA having a lower molecular weight can be separated from the cell culture by processing the pH adjusted broth through TFF filtration units in a high shear configuration, such as Minitan ® or Pellicon ® (Millipore) . Centrifugation or TFF filtration can be combined with size fractionation using conventional techniques such as filtration employing filter units having the desired molecular weight cut-off, or by size exclusion chromatography. For example, a filter unit having a 100,000 molecular weight cut-off is employed to separate ⁇ -PGA into lower and higher molecular weight fractions. In this manner, ⁇ -PGA of defined molecular weight and low poly dispersity is isolated from the high molecular weight polymer.
  • ⁇ -PGA can be concentrated in solution by employing TFF, whereby the molecular weight barrier of the tangential filtration device is smaller than the ⁇ -PGA molecular weight of interest .
  • ⁇ -PGA can be fractionated using a microfluidizer (Microfluidics, Massachusetts) cell disrupter.
  • the device is normally used to generate high sheer to disrupt bacterial cells.
  • the device is designed for continuous operation such that material from a reservoir is pumped into the device and processed effectively a fluid element at a time, the ⁇ -PGA solution is run through the unit for one pass only. Therefore, while each fluid element is being sheered or fractionated, the remainder of the solution is stable in the reservoir or in the collection vessel.
  • the device can be operated at a range of PSI. In one embodiment, the device is operated at a PSI of about 1,000 to about 20,000.
  • the method of the present invention includes increasing viscosity of a ⁇ -PGA solution.
  • a suitable chelator such as ethylenediamine tetra-acetic acid (EDTA) or EGTA is added to the ⁇ -PGA supernatant, or ⁇ -PGA filtrate, to remove metal ions bound to the ⁇ -PGA.
  • the ⁇ -PGA solution is dialyzed against an EDTA-containing solution at a concentration sufficient to remove divalent cations bound to the ⁇ -PGA.
  • the EDTA is at a concentration of between about 1 and about 10 M.
  • the concentration of EDTA is about 2 mM or higher.
  • the ⁇ -PGA solution is processed by diafiltration employing TFF and an EDTA-containing solution.
  • the diafiltration includes constant volume washing of ⁇ -PGA supernatant or ⁇ -PGA filtrate with an EDTA-containing solution.
  • the chelator is then removed from the solution by a suitable method, such as dialysis or diafiltration, against a suitable medium, such as water or an appropriate buffer and the ⁇ -PGA solution is lyophilized.
  • a suitable medium such as water or an appropriate buffer
  • an ammonium carbonate buffer can be used since, upon lyophilization, ammonia and C0 2 is liberated, leaving pure ⁇ -PGA as a fine white powder.
  • a buffer solution of high ionic strength is employed, dramatically reducing the fluid viscosity during this stage of processing, thereby decreasing processing time.
  • the buffer solution is at a concentration of least about 200 mM to about 400 mM.
  • processing of the ⁇ -PGA supernatant or ⁇ -PGA filtrate is facilitated by diluting the ⁇ -PGA solution to further reduce the viscosity.
  • removal of metal ions from ⁇ - PGA causes the ⁇ -PGA to have an increased capacity for heavy metal uptake.
  • the ⁇ -PGA having increased capacity for heavy metal uptake can be employed in environmental applications, such as water treatment to remove heavy metals .
  • the ⁇ -PGA of the invention includes ⁇ -PGA molecules of molecular weight greater than about 2.5 x 10 6 g/mol.
  • the ⁇ -PGA of the present invention has a molecular weight of about 4 to 8 x 10 6 g/mol. In other embodiments, the molecular weight can be at least about 8 x 10 6 or 10 x 10 6 .
  • ⁇ -PGA having a molecular weight of at least 1 x 10 8 has been purified using the method of the present invention.
  • the purified ⁇ -PGA preparation has less than about 8 ⁇ g of uronic acid per milligram of ⁇ -PGA.
  • the fractionated ⁇ -PGA of the present invention has a polydispersity of about 1.4 to 1.6.
  • Bacillus licheniformis ATTC#9945 The cells were grown in a seed broth consisting of 10 g/L peptone, 2 g/L yeast extract, and 0.02 g/L MgS0 4 -7H 2 0 (Kunioka and Gotoh, Applied Microbiology and Biotechnology, 40:1031-1035). The cells were grown overnight ( ⁇ 8 hr.) At 250 rpm and 37°C. A volume of 0.9 mL of the cells was then aseptically transferred to forty 1 mL cyrovials to which 0.1 mL of glycerol was added. The vials were then shaken vigorously and stored at -20°C. These vials were used to initiate all experiments.
  • the frozen cells were rapidly thawed in a temperature bath at 37°C and 50 mL of seed broth was inoculated with the thawed cells.
  • the inoculated seed broth (seed culture) was then grown at 250 rpm on a rotary shaker and 37°C.
  • the seed culture was then added aseptically to standard production medium at a volumetric ratio of 2%.
  • the production medium was a modified form of Medium E (Thorne et al .
  • Fermentation times ranged from 3 to 5 days.
  • the molecular weight range obtained medium was 2-3 x 10 6 Da, which corresponded to a viscosity (at zero shear) , for a 2 or 4% (w/v) solution at 25°C of 20,000 and 65,000 cP, respectively.
  • the yield of purified PGA was approximately 15 g/1.
  • viscosity of the fermentation broth was extremely high, and exhibited significant normal forces (similar to bread dough which causes the dough to "climb" up a mixer shaft) .
  • the viscosities obtained for 2 and 4% solutions at 25°C were 28,000 and as high as 150,000 cP. Since equivalent concentrations of material were involved, the only factor which could affect the viscosity under these conditions was molecular weight. Branching of the molecule could also affect viscosities, but no significant branching effect of the molecule was found.
  • the molecular weight of the ⁇ -PGA was 4 - 6 x 10 6 Da. The yield was 12 g/1. Control fermentations using medium developed by Thorne, et al , J. Bacteriology, 68:307-315 (1957) had a viscosity under the same conditions for analysis (2% solution at 25°C) of only 200 cP, however, the yield was as high as 40 g/1.
  • the fermentation of ⁇ -PGA producing bacteria cultured in the above described medium was performed in shake flasks and in bioreactors .
  • the conditions in the 4 liter bioreactors were the following: agitation speed - 200 RPM, temperature - 37°C and aeration - 5L/min.
  • Impellers used in the fermenter were three 6-bladed Rushton turbines. Similar molecular weight and viscosity was obtained in the fermenter.
  • Alternate Media Formula tions Alternate media formulations that yielded high molecular weight ⁇ -PGA are 1) production medium described under Example I that was pH adjusted with semi-conductor grade sodium hydroxide (99.99% pure), and 2) medium described under Example I, wherein prior to its addition, the citric acid monohydrate was dissolved in water and the pH of the citric acid solution adjusted to pH 6.0 with reagent grade sodium hydroxide. Al terna te Cryopreservation
  • Example II Cells were grown in production medium of Example II until an optimal density was obtained (typically exponential phase) .
  • An equal volume of cell culture and 20% glycerol were mixed and immediately stored at -80 °C in appropriate aliquots.
  • 1 ml of cryopreserved cells was thawed at room temperature and added to 50 ml of seed broth (described in Example I) . After 8 hours of incubation as described, the optical density of the culture at 600nm was approximately 1. Two ml at these exponential phase cells were inoculated into 50ml of production broth and incubated as described.
  • pH Adj ustmen t The culture was harvested by reducing the temperature of the fermentation broth to at least room temperature or cooler. The temperature was reduced because addition of acid has a tendency to increase the temperature, which increases the degradation rate of the polymer during processing. After cooling, the pH of the broth was reduced (using concentrated HCl) to approximately 2.0. Typically, 300 ml of concentrated HCl was used per 5 liters of culture. The material was then centrifuged at approximately 10,000 - 15,000 x g for 20 minutes. During centrifugation, a pellet forms which can be easily decanted. ⁇ -PGA produced via the low shear method described herein has ten-fold less contaminants (see Table 1) than was cited by McLean, et al . ⁇ Applied and Environmental Microbiology, 56:3617-3677).
  • the clarified broth (broth after cell removal) was dialyzed using 12,000 molecular weight cut-off dialysis tubing.
  • the first dialysis was performed against a 10 mM solution of EDTA.
  • the concentration of EDTA can be in the range of 1 mM to 100 mM.
  • MALLS Sta tic Mul tiple Angle Laser Light Scattering
  • the molecular weight of ⁇ -PGA prepared as described above (through Example III) was measured using SEC, a standard method in the art.
  • a TSK G ⁇ OOOPWxL column (ToSoHaaS, Montgomeryville, PA) was equilibrated with 0.4 M phosphate buffer (pH 4.5).
  • Polyethylene oxide of molecular weight 1 x 10 6 g/mol and 8 x 10 6 g/mol (PEOl and PE08, respectively) were used for comparison.
  • the molecular weight of ⁇ -PGA was well above 1 x 10 6 g/mol and very likely near 8 x 10 6 g/mol, in agreement with the static MALLS analysis.
  • Example VI Fractionating PGA during Purification
  • Fractionated ⁇ -PGA having lower molecular weight with low polydispersity was produced by filtering the pH- adjusted broth of Example 2 using a Minitan ® (Millipore) filtration device. The broth was circulated through the filter system such that only cleaved polymer passed through the filter, resulting in fractionated ⁇ -PGA of low polydispersity.
  • Minitan ® Micropore
  • ⁇ -PGA was prepared according to the method of the present invention and separated from the cells.
  • a ⁇ -PGA solution (up to 2% w/v) was prepared in deionized water and allowed to dissolve completely (overnight at 4°C) .
  • the ⁇ -PGA solution was processed through a Microfluidizer (Microfluidics, Massachusetts) cell disrupter. The device was used as described in the manufacturer' s instructions except using a cell-free solution. A pressure range of 1,000 up to 20,000 PSI was used.

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Abstract

L'invention concerne un η-PGA à masse moléculaire élevée. On sépare le η-PGA des cellules d'une culture de cellules productrice de η-PGA après avoir réglé le pH de la culture cellulaire à un niveau inférieur à 3 environ. De plus on augmente la viscosité de la solution η-PGA par le retrait d'ions métallique à l'aide d'un chélateur. Le η-PGA est en outre séparé par fractionnement directement dans une culture cellulaire de manière que ce η-PGA fractionné présente une faible polydispersité. On obtient ainsi un η-PGA purifié présentant une masse moléculaire d'au moins 2,5 x 106.
EP98960252A 1997-11-18 1998-11-18 Acide gamma-polyglutamique (gamma-pga) a masse moleculaire elevee Withdrawn EP1032699A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US6694297P 1997-11-18 1997-11-18
US66942P 1997-11-18
PCT/US1998/024580 WO1999025864A2 (fr) 1997-11-18 1998-11-18 ACIDE η-POLYGLUTAMIQUE (η-PGA) A MASSE MOLECULAIRE ELEVEE

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EP1032699A1 true EP1032699A1 (fr) 2000-09-06

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WO2004028548A2 (fr) * 2002-09-26 2004-04-08 Carbomer, Inc. Agents neuroprotecteurs
DE102004030938A1 (de) 2004-06-26 2006-01-12 Henkel Kgaa Neue, Polyaminosäuren bildende oder abbauende Genprodukte von Bacillus licheniformis und darauf aufbauende verbesserte biotechnologische Produktionsverfahren
WO2011127221A1 (fr) 2010-04-09 2011-10-13 National Health Research Institutes Solutions d'irrigation oculaire à base d'acide gamma-polyglutamique
CN103665371B (zh) * 2013-11-14 2016-03-02 天津北洋百川生物技术有限公司 一种利用超滤纳滤技术精制生物发酵液中聚谷氨酸的方法
CN109609408B (zh) * 2018-12-27 2022-04-15 黄河三角洲京博化工研究院有限公司 一株γ-聚谷氨酸高产菌株及利用该菌株进行液体发酵制备γ-聚谷氨酸的方法
CN112480394A (zh) * 2020-12-01 2021-03-12 广西大学 一种从高黏发酵液中分离纯化超高分子量聚γ-谷氨酸的方法

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