WO2001005949A1 - Procede de separation de membrane destine a purifier, a concentrer et a densifier les solutions d'enzymes avec des polyols - Google Patents

Procede de separation de membrane destine a purifier, a concentrer et a densifier les solutions d'enzymes avec des polyols Download PDF

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Publication number
WO2001005949A1
WO2001005949A1 PCT/EP2000/005908 EP0005908W WO0105949A1 WO 2001005949 A1 WO2001005949 A1 WO 2001005949A1 EP 0005908 W EP0005908 W EP 0005908W WO 0105949 A1 WO0105949 A1 WO 0105949A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
solution
polyol
enzymes
purifying
Prior art date
Application number
PCT/EP2000/005908
Other languages
English (en)
Inventor
Myongsuk Bae-Lee
Eric Charles Ehrnsperger
Feng-Lung Gordon Hsu
Kristina Marie Neuser
Original Assignee
Unilever N.V.
Unilever Plc
Hindustan Lever Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Unilever N.V., Unilever Plc, Hindustan Lever Ltd filed Critical Unilever N.V.
Priority to AU58202/00A priority Critical patent/AU5820200A/en
Publication of WO2001005949A1 publication Critical patent/WO2001005949A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass

Definitions

  • enzymes have increased in recent years through industrial, medical and domestic applications. Enzymes are naturally derived from several sources, such as plants, animals, bacteria, yeasts and fungi. Modern genetic engineering techniques have also widened the scope of 0 enzyme applications.
  • enzymes are useful for aiding in the removal of a variety of stains, as well as fabric care.
  • Suitable enzymes include, but are not 5 limited to, proteases, amylases, upases, cellulases, peroxidases and mixtures thereof.
  • the enzyme activity, concentration and density at the time of use or manufacture of product containing the 5 enzyme will typically govern how much of the enzyme, i.e. on a wt% basis, is used or placed in the product. For example, if the activity of an enzyme preparation is high, less amount of enzyme preparation is needed to ensure the desired level of total activity in the final product, as 0 compared to an enzyme preparation with a relatively lower level of activity (as discussed herein, 'enzyme preparation" includes constituents found in typical commercially available enzyme products such as, for example, enzymes, fermentation by-products, stabilisers, solvents, surfactants, and water) . Therefore, there is often a need for enzymes that can be supplied at higher levels of activity, concentration and/or density than previously available. Such control of enzyme preparations can lead to obvious production and shipping efficiencies and can lead to uses that would not be otherwise practicle or feasible.
  • Activity and storage stability of an enzyme preparation can be negatively influenced by impurities derived from fermentation process, such as, electrolytes, metals, peptides/proteins, carbohydrates, lipids, and nucleotides.
  • the impurities in the enzyme preparation can cause physical instability of the formulation and can also negatively affect the stability of other enzymes in the formulation. If one or more of these impurities can be decreased or eliminated from the enzyme preparation, there can be a corresponding benefit in terms of activity and/or stability.
  • Enzyme stability is important in order to deliver optimum enzymatic benefits.
  • the enzymes are often exposed to harsh or unnatural environments which can reduce the stability and activity of the enzymes.
  • Enzyme capsules for delivering the benefits of enzymes are also useful. Therefore, a physical barrier, such as a hydrophobic capsule containing the enzyme, can protect the enzyme from other agents that may have adverse affects on activity and stability.
  • current encapsulation technology is somewhat limited mainly due to enzyme density and enzyme loadings. Therefore, to achieve a desired density of capsule it would be advantageous to increase enzyme density without lowering enzyme loadings.
  • Enzyme capsules with hydrophobic barriers tend to have lower than desired density and tend to float in liquid formulations. By increasing enzyme density without diluting enzyme loadings, preparations can be delivered more efficiently and encapsulation technology would be greatly improved. It is also preferable to densify enzymes without concentrating impurities.
  • the present disclosure relates to methods and apparatuses for purifying, concentrating and/or densifying enzymes in solution (collectively ⁇ purifying" herein) .
  • the enzyme preparation is placed in at least partial contact with a polyol solution, such as sucrose, propylene glycol, glycerol and, more preferably, sorbitol or combinations of polyols.
  • a polyol solution such as sucrose, propylene glycol, glycerol and, more preferably, sorbitol or combinations of polyols.
  • the method allows for various impurities and water to leave the enzyme solution while leaving the enzymes behind and increasing polyol concentration in the enzyme solution.
  • the purification process not only purifies the enzyme solution, thereby enhancing stability, but it also concentrates and densifies the enzyme solution. Small scale and large scale apparati are discussed for carrying out this method.
  • Figure 1 illustrates a preferred apparatus for purifying, concentrating and/or densifying enzymes
  • Figure 2 illustrates an alternate apparatus for purifying, concentrating and/or densifying enzymes.
  • apparatus 10 illustrates a preferred method of purifying, concentrating and/or densifying an enzyme preparation.
  • Enzyme preparation 12 is disposed in partially permeable enclosure 14.
  • Enzyme preparation 12 can be any commercially available preparation or laboratory preparation.
  • Enclosure 14 is preferably fabricated from a membrane material that permits passage of molecules, other than the desired enzymes, therethrough. For example, if the molecular weight of the desired enzyme is about 20,000, a membrane that permits passage of molecules having a molecular weight less than 20,000 is desired.
  • An example of a suitable membrane is Spectra/Por® dialysis tubing (8,000 MWCO (molecular weight cut off) ) available from Spectrum, Website, Georgia Hills, CA. As shown, tubular enclosure 14 is closed at both ends by ties 16 to prevent leakage of enzyme preparation 12.
  • enclosure 14 containing enzyme preparation 12 is disposed in container 18.
  • Container 18 contains high density polyol solution 20 that can be circulated by mixer 22.
  • Liquid solution 20 is preferably prepared by dissolving at least one polyol in water. Most preferably, liquid solution 20 is a sorbitol solution.
  • the level of polyol in solution will affect the rate of exchange of molecules between the solution and the enzyme preparation. Between about 50% to about 80% sorbitol provides a satisfactory rate of transfer, however higher or lower levels can be used depending on desired density of enzyme solution.
  • Enzyme activity of protease and lipase was measured with a standard enzyme using casein and p-nitrophenylvalerate, respectively, as a substrate.
  • 15 - contact time a couple of hours to days depending on desired density, activity and impurity level
  • Vessel 102 contains raw enzyme preparation 200 that can be pumped into enzyme reactor 110 by pump 108 through valves 104 and 106. Processed enzyme preparation
  • Vessel 122 contains sugar solution 300 that can be pumped into lower portion 130 of reactor 110 by pump 126 through valves 124 and 128.
  • Solution 300' that which has removed components from enzyme solution 200, is removed through valve 128 by pump 134 and can be recycled or discarded.
  • membrane 114 is any suitable membrane that prevents or inhibits desired enzymes from passing into lower portion 130 of reactor 110 while allowing impurities or other undesirable molecules to pass into lower portion 130.
  • Mixers 112 and 132 are provided to ensure sufficient flow on each side of membrane 114.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Sustainable Development (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des procédés et des appareils de purification, de densification et/ou de concentration d'enzymes. Dans un mode de réalisation préféré, une préparation d'enzymes est mise en contact avec une solution d'eau et de polyol, éliminant ainsi les impuretés contenues dans la préparation d'enzymes et augmentant la densité et/ou la concentration de ces enzymes.
PCT/EP2000/005908 1999-07-15 2000-06-26 Procede de separation de membrane destine a purifier, a concentrer et a densifier les solutions d'enzymes avec des polyols WO2001005949A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU58202/00A AU5820200A (en) 1999-07-15 2000-06-26 Membrane separation process for purifying, concentrating and densifying enzyme solutions with polyols

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14401499P 1999-07-15 1999-07-15
US60/144,014 1999-07-15

Publications (1)

Publication Number Publication Date
WO2001005949A1 true WO2001005949A1 (fr) 2001-01-25

Family

ID=22506691

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/005908 WO2001005949A1 (fr) 1999-07-15 2000-06-26 Procede de separation de membrane destine a purifier, a concentrer et a densifier les solutions d'enzymes avec des polyols

Country Status (2)

Country Link
AU (1) AU5820200A (fr)
WO (1) WO2001005949A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6420333B1 (en) 2001-08-28 2002-07-16 Unilever Home & Personal Care Usa Division Of Conopco, Inc. Manufacture of capsules for incorporation into detergent and personal care compositions
US6730651B2 (en) 2001-08-28 2004-05-04 Unilever Home & Personal Care Usa Division Of Conopco. Inc. Concentrated stock of capsules for detergent or personal care compositions
EP3165604A1 (fr) * 2015-11-05 2017-05-10 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de purification de compositions de virus et de telles compositions obtenues
WO2017076553A1 (fr) * 2015-11-05 2017-05-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé pour la séparation de compositions virales comprenant l'appauvrissement et la purification de ces dernières

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001366A1 (fr) * 1989-07-14 1991-02-07 Biosphere Corporation S.A. Procede de stabilisation de bacteries par centrifugation en une pate non aqueuse
JPH04304886A (ja) * 1991-03-29 1992-10-28 Takara Shuzo Co Ltd 制限酵素の製造方法
US5354669A (en) * 1992-08-12 1994-10-11 Boehringer Mannheim Gmbh Type II restriction endonuclease SexAI

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001366A1 (fr) * 1989-07-14 1991-02-07 Biosphere Corporation S.A. Procede de stabilisation de bacteries par centrifugation en une pate non aqueuse
JPH04304886A (ja) * 1991-03-29 1992-10-28 Takara Shuzo Co Ltd 制限酵素の製造方法
US5354669A (en) * 1992-08-12 1994-10-11 Boehringer Mannheim Gmbh Type II restriction endonuclease SexAI

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 199250, Derwent World Patents Index; Class B04, AN 1992-409779, XP002151340 *
DATABASE WPI Section Ch Week 199549, Derwent World Patents Index; Class D16, AN 1995-381207, XP002151341 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6420333B1 (en) 2001-08-28 2002-07-16 Unilever Home & Personal Care Usa Division Of Conopco, Inc. Manufacture of capsules for incorporation into detergent and personal care compositions
US6730651B2 (en) 2001-08-28 2004-05-04 Unilever Home & Personal Care Usa Division Of Conopco. Inc. Concentrated stock of capsules for detergent or personal care compositions
EP3165604A1 (fr) * 2015-11-05 2017-05-10 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de purification de compositions de virus et de telles compositions obtenues
WO2017076553A1 (fr) * 2015-11-05 2017-05-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé pour la séparation de compositions virales comprenant l'appauvrissement et la purification de ces dernières
US11697800B2 (en) 2015-11-05 2023-07-11 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Method for the separation of virus compositions including depletion and purification thereof

Also Published As

Publication number Publication date
AU5820200A (en) 2001-02-05

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