WO2003076456A2 - Procede de recuperation de proteines par precipitation - Google Patents

Procede de recuperation de proteines par precipitation Download PDF

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Publication number
WO2003076456A2
WO2003076456A2 PCT/EP2003/002603 EP0302603W WO03076456A2 WO 2003076456 A2 WO2003076456 A2 WO 2003076456A2 EP 0302603 W EP0302603 W EP 0302603W WO 03076456 A2 WO03076456 A2 WO 03076456A2
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WO
WIPO (PCT)
Prior art keywords
protein
solution
proteins
enzyme
precipitate
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.)
Ceased
Application number
PCT/EP2003/002603
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English (en)
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WO2003076456A3 (fr
Inventor
Mylene Caussette
Veronique Ferreol
Brigitte Lindet
Gabriel Marinus Henricus Meesters
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DSM IP Assets BV
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DSM IP Assets BV
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Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Priority to AU2003212348A priority Critical patent/AU2003212348A1/en
Publication of WO2003076456A2 publication Critical patent/WO2003076456A2/fr
Publication of WO2003076456A3 publication Critical patent/WO2003076456A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation

Definitions

  • the present invention relates to a method for purification or separation of a protein from a solution containing dissolved proteins.
  • Proteins that are used in the food industry are most often produced fermentatively in a microbial culture. These fermentatively produced proteins are in most cases used as a raw enzyme extract, i.e. in unpurified form, since the costs of protein purification are often too high.
  • the fermentation broth may be filtered, with the aim to separate the cells from the proteins, eliminate some of the small molecules in solution and concentrate the macromolecules such as the proteins.
  • the extract thus obtained comprises the desired protein, and additionally other proteins and macromolecules (polyphenols, colorants, etc.), which are produced by the cells or which have been introduced into the culture medium.
  • the raw enzyme extract therefore has a very undefined composition due to its complexity.
  • Proteins may be recovered from a protein containing solution by precipitation in a crystalline form.
  • EP 0 506 866 discloses a method for the crystallisation of proteins wherein a crystallisation agent, such as Na, K, Ca or Mg formate, acetate or nitrate is added.
  • a crystallisation agent such as Na, K, Ca or Mg formate, acetate or nitrate
  • Another way to crystallise proteins in a protein-containing aqueous solution is the treatment of the solution with a water-miscible organic solvent (WO 97/34919) in an amount sufficient to bring about crystallization.
  • WO 97/34919 water-miscible organic solvent
  • the addition of an organic solvent to recover a protein for use in the food industry is not desirable.
  • US 4,659,667 discloses a process for the crystallisation of proteins using evaporation of water under vacuum. Nevertheless, industrial evaporation processes are commonly very expensive, and are thus less attractive for application in the food industry.
  • An alternative crystallisation process for the recovery of a protein from a protein- containing solution has been disclosed in US 6,066,481. Here the protein solution is treated with a salt containing a sulphur atom having an oxidation state less than 6, and recovering the protein in crystalline form.
  • a protein can be recovered and partially purified from a solution containing dissolved proteins by the method of the present invention, wherein a protein is recovered from a solution containing dissolved proteins by treating said solution with a chaotropic salt, which comprises
  • the solution is preferably an aqueous solution.
  • Chaotropic salts are known to increase the solubility of non polar substances in water. They can also denature proteins because they have the ability to disrupt hydrophobic interactions.
  • Chaotropic salts that may be used in the present invention are for example salts of perchlorate (CIO ), thiocyanate (SCN ), hydrogensulfate (HSO 4 " ), dihydrogenphospate (H 2 PO 4 " ), hydrogencarbonate (HCO 3 " ), iodide (I “ ), chloride (Cl “ ), nitrate (NO 3 " ), guanidinium chloride, urea or trichloroacetate.
  • Sulphate for example amonium sulphate, is not according to our definition a chaotropic salt but an kosmotropic salt or lyotropic salt.
  • the chaotropic salt is a thiocyanate (SCN “ ) or perchlorate (CLO 4 " ).
  • the thiocyanate salt is a sodium or a potassium salt.
  • the thiocyanate salt may be added in solution or in an anhydrous form in an amount effective to precipitate a protein from the solution containing dissolved proteins.
  • the final concentration of thiocyanate in the solution containing dissolved proteins is at least 0.01 M, more preferably at least 0.02 M, and most preferably at least 0.03 M and/or the final concentration of thiocyanate in the solution containing dissolved protein is preferably lower than 2M, more prefarably lower than 1 M and most preferably lower than 0.5M.
  • chaotropic salts for the other chaotropic salts also concentrations of between 0.01 M and 2M are also generally used.
  • concentrations of between 0.01 M and 2M are also generally used.
  • the effect of thiocyanate to precipate protein according to invention can be enhanced by the addition of suitable amounts of sulphate to increase the yield of precipate.
  • the protein may precipitate as an amorphous solid or as a crystal.
  • An amorphous solid is defined as a solid of which the protein molecules have a diffuse structure.
  • a crystal is defined as a solid having a regularly repeating internal arrangement of its protein molecules. Both the amorphous form and the crystalline form of the protein may be present at the same time in the precipitated product.
  • the method of the invention results in a precipitated protein which is more pure (in wt% dry matter) than the protein in the starting composition. In this way it becomes possible to purify a protein from for example a fermentation broth whereby the protein has preferably a purity of at least 80 wt%, more preferably of at least 90 wt% on dry matter.
  • the fermentation broth can be pretreated, for example be filtered or centrifuged in order to separate biomass or other unwanted components from the broth.
  • the present invention also provides a method wherein at least two proteins are separately precipitated from the solution containing the at least two dissolved proteins.
  • conditions are chosen wherein in first instance the first protein is precipitated.
  • the conditions can be amended to precipitate the second protein.
  • the first precipitate will be removed before the second protein is precipitated.
  • the precipitates can be separated afterwards in a fraction containing the precipitate of the first protein and a fraction of the precipitate of the second fraction. For example difference in particle size, density of the crystals and re-dissolvability can be used to separate the proteins.
  • Quick precipitation generally results in a combination of a crystaline and amorphous precipitate.
  • the precipitate is in a form of a slurry containing the solid product.
  • This slurry can be dried after which the solids may be granulated.
  • the slurry can be used as the binder containing liquid needed for granulation.
  • the slurry contains a high concentration or protein which can be dissolved again in solvents like glycerol ar sorbitol. These solvents can be used as the formulation liquid which stabilizes the product microbially as well as stabilizes the protein itself. This process saves a substantial amount of glycerol and sorbitol compared to other processes for example ultra filtration (UF), which require more water and yield a lower concentration of protein.
  • UF ultra filtration
  • the aqueous solution containing dissolved proteins as defined in the method of the present invention may be any solution comprising more than one protein.
  • the solution containing dissolved proteins is obtained from a fermentation broth.
  • Such a fermentation broth may be obtained by fermentation of a microorganism, for example a bacterium or a fungus in any suitable nutrient medium.
  • a protein that is recovered by the method of the present invention from a solution containing dissolved proteins may be a protein such as an enzyme which is used in any industry, for example the food, pharmaceutical, chemical, analytical or detergent industry.
  • a solution containing dissolved proteins in this regard may be a fermentation broth, or a cell culture and can be used for the production of pharmaceutical products.
  • dissolved proteins is meant a solution containing the protein which is a clear or cloudy solution, whereby the clear solution is preferred, so the protein is preferably completely dissolved.
  • not completely dissolved protein, such as a suspension, or protein dissolved using a dissolving improving agent such as a detergent are part of the present process.
  • the precipitated protein is an enzyme.
  • the enzyme may be any enzyme used in the food, agricultural, pharmaceutical or detergent industry, such as a protease, a lipase, a pectinase, an amylase, a phytase, a cellulase, or a xylanase.
  • Suitable enzymes to be precipitated according to method of the present invention include any enzyme that may be a fermentation product from a cell such as a microorganism.
  • the enzyme is from microbial origin, more preferably the enzyme is from fungal origin. Chemically or genetically modified mutants are included.
  • the enzyme that is precipitated according to the method of the present invention is an acid enzyme.
  • the pH at which the activity of the enzyme is optimal and the pH at which the enzyme is stable ranges between pH 1 and 7, more preferably between pH 2 and 5.
  • proteases to be precipitated according to the method of the present invention are acid proteases, such as Fromase®, an enzyme extract produced by DSM, Marzyme® produced by Rhodia, Microlant TM produced by Chr. Hansen, and Valiren® produced by Valley Research. These commercial preparations comprising an acid protease, which are all produced by Rhizomucor miehei, are used in the cheese-making industry for coagulation of milk.
  • the Rhizomucor miehei protein extract may comprise, besides an acid protease, a ⁇ -glucanase, an esterase-lipase, and sometimes amylase as side activities.
  • Another acid protease is chymosin which can be produced fermentatively in for example K. lactis or A. niger.
  • precipitation occurs at a pH below the isoelectric point of the protein to be precipitated.
  • the precipitation occurs at a pH between 1 and 4, more preferably between 1.5 and 3.8.
  • the pH of the solution containing dissolved proteins in which precipitation of a protein occurs may be adjusted to the desired pH by the addition of any suitable acid or buffer solution.
  • a suitable acid may be phosphoric acid or hydrochloric acid.
  • a suitable buffer solution may be a phosphate-citrate buffer solution.
  • the temperature at which precipitation of a protein in a solution containing dissolved proteins occurs is between 2°C and 50°C. Preferably, said temperature is between 3°C and 30°C.
  • the protein precipitated by the method of the current invention can be recovered by .standard methods known to a person skilled in the art.
  • Y m (mass of recovered protein)/(mass of initial protein) * 100 (%)
  • the protein content of the starting sample and end product sample is determined (Lowry).
  • An electrophoresis on agarose gel (Sebia gel HR) is then performed on both samples in order to determine the ratio of the different proteins present in the solution. From this ratio, the mass of each protein (for example enzyme) can be calculated. Yield based on enzyme activity
  • a solution is made containing a commercially available chymosin (Maxiren®, 8 g/l chymosin) and 0.04M sodium thiocyanate. This solution is kept at pH 2.5 and 5°C.
  • a solution is made containing 20-25% W/W endo-1 ,3 (4)- ⁇ -glucanase of the total protein content and containing sodium thiocyanate within the range 0-0.15M is kept at pH 3.0 and 5°C.
  • crystals of the pure enzyme 99% w/w of total protein
  • the crystals were centrifuged and lyophilised.
  • the crystals have a needle morphology of a 100 ⁇ m size, as determined by transmission electronic microscopy.
  • endo-1 ,3 (4) ⁇ -glucanase determined as follows: 2 ml of the enzyme solution was added to 15 ml of a 1 % (v/v) solution of barley cream (Moulin Waast, Mons en Pevele) at pH 5.6 and at 45°C. The decrease in viscosity was determined in Hubbelhode no° 1 C viscosimeter, during 15 min.
  • One unit of BGF endo-1 ,3 (4)- ⁇ - glucanase is defined as the enzymatic activity which will lead to a variation of viscosity with a speed which constant is 0.147 min-1 for 1 ml of medium in the assay conditions.
  • Table 1 shows the Y m and Y e as a function of the initial thiocyanate concentration and as a function of the initial protein concentration.
  • Table 1 shows that both the initial protein concentration and the initial thiocyanate concentration have an effect on the yields. Yield higher than 80% can be achieved.
  • Table 2 shows the effect of the pH on the crystallisation of endo-1 ,3 (4)- ⁇ -glucanase. pH stability range of the enzyme (pH range 2.5-7.5) with a fixed sodium thiocyanate concentration of 0.04M (protein concentration 16 g/l).
  • Table 2 shows that no crystals are formed above pH 4. The crystals are only formed below the isoelect c point of enzyme (pH 4.2). Both tables show that there is no denaturation of the enzyme (Y m is comparable to Y e ).
  • Table 3 shows that an increased sulfate concentration (0-2 M) results in an increase of the Y m of the enzyme crystallisation process with sodium thiocyanate.
  • a solution is made containing Fromase® (containing both ⁇ -glucanase and protease), protease being present in the solution 60-67.5 g/l, and thiocyanate (0.04M).
  • the solution is incubated for one day at pH 3 and 5°C.
  • the ⁇ -glucanase is first recovered.
  • the thiocyanate concentration is increased to 0.1 M.
  • This solution is kept at pH 3 and 5°C. After less than 24 hours of incubation, precipitation occurs.
  • the Y m and the Y e were both 19%, which indicated that no denaturation of the protein had occurred.
  • the example shows that the separation of two enzymes is possible.
  • protease The activity of protease was measured as described by the International Dairy Federation (IDF); protocol 157:1992 and can be expressed in IMCU (International Milk Clotting Unit).
  • IDF International Dairy Federation
  • IMCU International Milk Clotting Unit

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de récupérer une protéine à partir d'une solution contenant des protéines dissoutes, par traitement de ladite solution avec un sel chaotropique. Ce procédé comprend les étapes consistant: à ajouter le sel chaotropique à la solution contenant des protéines en quantité suffisante pour précipiter la protéine; et à récupérer ladite protéine sous forme de précipité.
PCT/EP2003/002603 2002-03-12 2003-03-07 Procede de recuperation de proteines par precipitation Ceased WO2003076456A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003212348A AU2003212348A1 (en) 2002-03-12 2003-03-07 Hepatitis c virus replicon containing a reporter gene and a selectable marker gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02076340 2002-03-12
EP02076340.5 2002-03-12

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WO2003076456A2 true WO2003076456A2 (fr) 2003-09-18
WO2003076456A3 WO2003076456A3 (fr) 2003-12-24

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087016A1 (fr) * 2004-03-15 2005-09-22 Technische Universiteit Delft Technique de preparation d'un agregat de proteines et preparation pharmaceutique
WO2008065138A1 (fr) * 2006-11-29 2008-06-05 Novo Nordisk A/S Nouveau cristal d'insuline et son procédé de préparation
WO2014187983A1 (fr) 2013-05-24 2014-11-27 Commissariat à l'énergie atomique et aux énergies alternatives Procédé pour caractériser par spectrométrie de masse en tandem un échantillon biologique

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5523231A (en) * 1990-02-13 1996-06-04 Amersham International Plc Method to isolate macromolecules using magnetically attractable beads which do not specifically bind the macromolecules
EP0796335A1 (fr) * 1994-12-07 1997-09-24 Bionebraska, Inc. Production de peptides au moyen de proteines de fusion de recombinaison
US20030048341A1 (en) * 2000-09-25 2003-03-13 Mutz Mitchell W. High-throughput biomolecular crystallization and biomolecular crystal screening

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005087016A1 (fr) * 2004-03-15 2005-09-22 Technische Universiteit Delft Technique de preparation d'un agregat de proteines et preparation pharmaceutique
US7777012B2 (en) 2004-03-15 2010-08-17 Technische Universiteit Delft Method of preparing a protein aggregate and a pharmaceutical preparation
WO2008065138A1 (fr) * 2006-11-29 2008-06-05 Novo Nordisk A/S Nouveau cristal d'insuline et son procédé de préparation
WO2014187983A1 (fr) 2013-05-24 2014-11-27 Commissariat à l'énergie atomique et aux énergies alternatives Procédé pour caractériser par spectrométrie de masse en tandem un échantillon biologique

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AU2003212348A8 (en) 2003-09-22
WO2003076456A3 (fr) 2003-12-24

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