WO2011131594A1 - Immobilisation d'enzymes par utilisation de plasmas - Google Patents

Immobilisation d'enzymes par utilisation de plasmas Download PDF

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Publication number
WO2011131594A1
WO2011131594A1 PCT/EP2011/056069 EP2011056069W WO2011131594A1 WO 2011131594 A1 WO2011131594 A1 WO 2011131594A1 EP 2011056069 W EP2011056069 W EP 2011056069W WO 2011131594 A1 WO2011131594 A1 WO 2011131594A1
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WO
WIPO (PCT)
Prior art keywords
plasmas
enzymes
plasma
enzyme
plastic
Prior art date
Application number
PCT/EP2011/056069
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German (de)
English (en)
Inventor
Uwe Bornscheuer
Karsten Schroeder
Ulf Menyes
Dominique Boettcher
Torge Vorhaben
Dagmar Jasinski
Marko Haeckel
Original Assignee
Neoplas Gmbh
Institur Für Biochemie Der Ernst-Moritz-Arndt-Universität Greifswald
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 Neoplas Gmbh, Institur Für Biochemie Der Ernst-Moritz-Arndt-Universität Greifswald filed Critical Neoplas Gmbh
Publication of WO2011131594A1 publication Critical patent/WO2011131594A1/fr

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    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds

Definitions

  • the invention relates to a method for the treatment of plastic surfaces with cold plasmas (low temperature plasmas) and subsequent immobilization of enzymes on the treated surfaces to increase the enzyme activity and the stability of application of immobilized enzymes and an enzyme-carrier system obtainable by this method.
  • Enzymes have previously been bound by a combination of physical and chemical techniques on plastic surfaces. Since plastics usually have hydrophobic surface properties, but enzymes are usually provided as peptides from aqueous solutions for immobilization, the plastic surface must first be hydrophilized. This is done according to Bosley et al. JAOCS 74_ 107-11 1 (1997) frequently by wetting with a water-soluble alcohol as a wetting agent. Another approach is treatment with physical plasmas using spacer-forming nitrogen-containing compounds having carbon chains, such as amines to the plasma gases.
  • an inorganic oxide layer preferably of plasma-deposited organosilicon compounds, is additionally introduced between the polymer layer and the plastic carrier in order to promote adhesion.
  • azalactone or azalactone polymer combinations are used as the coupling agent using high energy radiation to generate surface radicals.
  • disulfite functions are used for the coupling of polypeptides on carriers using radiation. Fields of application of these systems are molecular biology, biochemistry, pharmacology and medical diagnostics.
  • Patent DE 69737654 T2 includes the use of plasma radiation for crosslinking, ethylene glycol bis (succinimidyl succinate) preferably being used here as an additional crosslinking agent.
  • the prior art also includes the publication of YIN, Yongbai, et al: "Plasma Polymer Surface Compatible with a CMOS Process for Direct Covalent Enzyme Immobilization.”, Plasm. Process. Polym. 2009, 6, 68-75. It describes a process for the covalent attachment of enzymes to plasma polymerized materials for sensor systems.
  • the paper discloses the use of the plasma polymers for CMOS sensors (i.e., complementary metal oxide semiconductor). In doing so, plasma coatings (e.g., acetylene) are deposited on metals.
  • CMOS structures are well suited for sensor technology, but not for use in chemical processes in industrial biocatalysis.
  • Plasma polymer coatings are called, i. Sensor surfaces are subsequently provided with active plasma-polymerized layers.
  • the enzymes mentioned are peroxidases and catalases.
  • the disadvantage consists partly in the complex plasma processes used and in the also complicated subsequent chemical steps for coupling the enzymes to the surface.
  • the aim is obviously an increase in stability for the resulting enzyme-carrier combinations.
  • economical implementation of these production processes is only possible if the enzyme-carrier combinations thus obtained are used in applications with high margins per unit of product produced therewith. This usually only applies to pharmaceutical products.
  • the use of such elaborately prepared enzyme-carrier systems is generally not given.
  • a major disadvantage of the publication by Nosworthy et al. is that the surface is only fissured by ion bombardment (at least 50 nm deep) and not highly activated by the PIII method.
  • the nitrogen in the Plll process leaves no nitrogen-functional groups on the surface, but in the "subsurface", ie near-surface regions (typically 20 nm below the surface), which are not accessible for hydrophilization (penetration depth of the enzyme solution is here less than 1 nm).
  • the invention had the object to eliminate the disadvantages of the technical solutions described in the prior art.
  • the preferably used commercially available plastic carrier materials are treated by means of physical cold plasmas from gas discharge processes, the plasma treatment taking place without spacer-forming, nitrogen-containing compounds with carbon chains.
  • spacer-forming nitrogen-containing organic compounds having carbon chains is meant, in accordance with the teachings of this invention, chemical compounds containing a carbon-nitrogen compound, such as amines or amides, for the purpose of forming nitrogen-functionalized surfaces.
  • these low-temperature plasmas are discharges in gases, which frequency technology by means of high-energy radio in the range low-frequency to high-frequency waves to microwaves are generated.
  • plasmas can be used both in vacuum reactors in the low pressure range and in the normal pressure range, e.g. operated with jet, barrier and hollow cathode discharges.
  • gas discharge is used in which there is a high density of charge carriers per unit volume of gas, the gas temperature being limited by suitable process control in order to avoid thermal modification of the material.
  • the surface of the plastics for treatment and immobilization with enzymes preferably from the class of hydrolases, preferably carboxylesterases, particularly preferably esterases or lipases and the class of oxygenases, preferably oxidoreductases, particularly preferably monooxygenases, especially preferably Baeyer-Villiger monooxygenases, optimally changed.
  • the high technical complexity of the enzyme coupling, as described in the prior art, for enzymes from the class of esterases, lipases and oxigenases circumvent, by the appropriate choice of a carrier, preferably a polyalkene carrier such Polypropylene in combination with a simple rapid plasma treatment, which achieves a hydrophilization of the support surface of less than 60 degrees for the water contact angle, preferably less than 10 degrees.
  • the plasma gases used are not plasma-polymerizable gases, preferably argon or argon / oxygen or argon / air mixtures. It can also contain nitrogen-containing gases such as ammonia.
  • the supports can be used in the form of plates, nets, membranes, other structures or preferably as a powder, porous or non-porous.
  • the immobilization of the enzymes is carried out from solutions with and without buffer by storing in the solution for at least 1 min to several hours or days preferably 8-16 hours with and without mixing. Preferably followed by washing and drying of the enzyme-carrier complex. A direct use without washing and drying is also possible.
  • the plasma treatment according to the invention produces special "highly activated" polymer surfaces which are functionalized to a maximum depth of 3 nm and thereby avoids the fracturing of the surfaces in contrast to the prior art, where the surface is fissured by targeted ion bombardment from the plasma.
  • microwave plasmas in the GHz range preferably by 2.54 GHz, or radio frequency plasmas (1 kHz - 100 MHz) in the low pressure or atmospheric pressure range to maximum densities of reactive species such as radicals and ions for the high activation of the surface to lead.
  • the following conditions are achieved in the plasma: RF plasmas are ignited with powers of 1 W to 100 W microwave plasmas of 100 W to 1500 W at gas flow rates of 1 to 100 sccm.
  • the ions are accelerated with voltages of maximum 500V. Under these conditions, the substrates are treated for 0.5 second to 600 seconds.
  • Annealing processes which are carried out at 350-400 ° C, are among the
  • Plasma uptake activations according to the invention are not necessary
  • an enzyme-carrier system is provided from a treated with physical gas discharge plasmas plastic carrier, preferably from a polyalkene polymer such as polypropylene, but also others Plastics without the addition of auxiliary components in the plasma gas used, which should serve in the prior art as a spacer for covalent bonding of the enzymes and enzymes, preferably from the class of esterases, lipases and oxygenases.
  • plastic carrier preferably from a polyalkene polymer such as polypropylene, but also others Plastics without the addition of auxiliary components in the plasma gas used, which should serve in the prior art as a spacer for covalent bonding of the enzymes and enzymes, preferably from the class of esterases, lipases and oxygenases.
  • no additional excipient for the immobilization of the enzymes is used on the support surface, which would have the purpose of providing a chemical bond between plasma-treated carrier and the enzyme.
  • a significant increase in the enzyme activity of the enzyme carrier systems is achieved when using the plasma-treated plastic carrier compared to the untreated carriers. Furthermore, an improvement of the storage and use stability is achieved.
  • the polypropylene powder is treated in an RF plasma system at 27.2 MHz.
  • 5 g of the powder are fluidized before the plasma treatment on a 50 Hz vibrating stainless steel sample holder in oxygen at 0.1 mbar for 30 min.
  • the plasma treatment is carried out in oxygen plasma at 80 W and a pressure of 0.1 mbar for 5 min.
  • the sample is stored at room temperature prior to enzyme treatment.
  • the polypropylene powder is treated in an RF plasma system at 27.2 MHz.
  • 5 g of the powder are fluidized before the plasma treatment on a 50 Hz vibrating stainless steel sample holder in oxygen at 0.1 mbar for 30 min.
  • the plasma treatment is carried out in oxygen plasma at 100 W and a pressure of 0.1 mbar for 1 s.
  • the sample is stored at room temperature prior to enzyme treatment.
  • Enzyme immobilization using the example of CalB lipase 500 mg of the plasma-treated polypropylene powder is incubated in a 15 ml glass jar with 4 ml of enzyme solution. For this purpose, a 10 mM sodium phosphate buffer at pH 7 is used. The incubation is carried out at 20 ° C overnight with a stirring speed of 200 revolutions / min. After incubation, the material is filtered off, washed twice with the buffer used for the immobilization (see above) and dried overnight in vacuo. Storage before the activity measurements takes place at 4 ° C.
  • the determination of the activity by means of the pH-stat method. These automatic Titrationsstrom (Titroline alpha ®, Schott, Germany) was used. For the determination, 25 ml of an emulsion of 5% (w / v) tributyrin and 2% (w / v) gum arabic in distilled water is used. A known amount of immobilized CalB is added to the emulsion and the release of the acid is determined titrimetrically by the addition of 10 mM NaOH at 37 ° C and a constant pH of 7.5. A tributyrin unit (TBU) is defined as the release of 1 ⁇ butyric acid per minute by the immobilized enzyme.
  • TBU tributyrin unit
  • the plasma treatment of 1.5 g of the polypropylene powder is carried out in a commercially available microwave plasma reactor at 2.45 GHz.
  • the system is purged with argon and then at a pressure of 1 mbar at 1200 W and a gas composition of 60/40 (v / v) oxygen / argon for 10s effective plasma treatment time in the pulsed plasma with a pulse rate of 10/90 on / off and a pulse rate of 10 Hz.
  • the treated material is stored at room temperature.
  • the plasma treatment of 1.5 g of the polypropylene powder is carried out in a commercially available microwave plasma reactor at 2.45 GHz.
  • the system is purged with argon and then at a pressure of 0.1 mbar at 1500 W and a gas composition of 80/20 (v / v) oxygen / argon for 100 ms (100 milliseconds) effective plasma treatment time in the pulsed plasma with a pulse rate of 10/90 on / off and a pulse frequency of 1 kHz treated.
  • the treated material is stored at room temperature.
  • 500 mg of the plasma-treated polypropylene powder is incubated in a 15 ml glass vessel with 4 ml of enzyme solution.
  • a 10 mM sodium phosphate buffer at pH 7 is used.
  • the incubation is carried out at 20 ° C overnight with a stirring speed of 200 revolutions / min.
  • the material is filtered off, washed twice with immobilization buffer and dried overnight in vacuo. Storage before the activity measurements takes place at 4 ° C.
  • the determination of the activity is carried out by means of a rapid test in 24-well microtiter plates. To this is added 400 ⁇ l of a solution of tributyrin in DMSO (10 mg / ml) to 1.2 ml of sodium phosphate buffer (5 mM, pH 7.3) and the solution stirred at room temperature. After addition of 400 ⁇ bromothymol blue (1:10 (v / v) dissolved in DMSO), a defined amount of the immobilized enzyme is added. Butyric acid released by the enzyme reaction changes the color of the solution from blue to yellow. The speed of the color change gives an indication of the activity of the material. For an accurate determination of the activity, the pH-stat method (see Example 1) is used.

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  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un procédé de traitement de surfaces en matière plastique au moyen de plasmas froids (plasmas à basse température), avec immobilisation subséquente d'enzymes sur les surfaces traitées, caractérisé en ce que le traitement au plasma conduit à une haute activation de la surface en matière plastique à une profondeur maximale de 3 nm, et en ce que le traitement au plasma s'effectue sans composés espaceurs contenant de l'azote, à chaînes carbonées, et en ce qu'on utilise des plasmas micro-ondes dans un domaine Ghz ou des plasmas radiofréquence (1 kHz - 100MHz) en atmosphère basse pression ou sous pression atmosphérique, pour haute activation de la surface en matière plastique.
PCT/EP2011/056069 2010-04-19 2011-04-16 Immobilisation d'enzymes par utilisation de plasmas WO2011131594A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010027936A DE102010027936A1 (de) 2010-04-19 2010-04-19 Immobilisierung von Enzym unter Verwendung von Plasmen
DE102010027936.6 2010-04-19

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WO2011131594A1 true WO2011131594A1 (fr) 2011-10-27

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4757014A (en) 1985-11-08 1988-07-12 Minnesota Mining And Manufacturing Company Immobilization of biologically active protein on a polymeric fibrous support
US5344701A (en) 1992-06-09 1994-09-06 Minnesota Mining And Manufacturing Company Porous supports having azlactone-functional surfaces
WO1995034814A1 (fr) * 1994-06-13 1995-12-21 Abbott Laboratories Traitement au plasma de matieres polymeres afin d'ameliorer l'immobilisation d'analyte sur celles-ci
WO2003082483A1 (fr) * 2002-03-28 2003-10-09 Plasso Technology Limited Preparation de revetements au moyen de la polymerisation par plasma
WO2007000163A1 (fr) 2005-06-27 2007-01-04 Bionanophotonics A/S Immobilisation de polypeptides par irradiation
DE69737654T2 (de) 1996-06-03 2007-12-27 Gore Enterprise Holdings, Inc., Newark Materialien und verfahren zur immobilisierung von bioaktiven substanzen auf biologisch abbaubare polymere

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4757014A (en) 1985-11-08 1988-07-12 Minnesota Mining And Manufacturing Company Immobilization of biologically active protein on a polymeric fibrous support
US5344701A (en) 1992-06-09 1994-09-06 Minnesota Mining And Manufacturing Company Porous supports having azlactone-functional surfaces
WO1995034814A1 (fr) * 1994-06-13 1995-12-21 Abbott Laboratories Traitement au plasma de matieres polymeres afin d'ameliorer l'immobilisation d'analyte sur celles-ci
DE69737654T2 (de) 1996-06-03 2007-12-27 Gore Enterprise Holdings, Inc., Newark Materialien und verfahren zur immobilisierung von bioaktiven substanzen auf biologisch abbaubare polymere
WO2003082483A1 (fr) * 2002-03-28 2003-10-09 Plasso Technology Limited Preparation de revetements au moyen de la polymerisation par plasma
WO2007000163A1 (fr) 2005-06-27 2007-01-04 Bionanophotonics A/S Immobilisation de polypeptides par irradiation

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALVAREZ ET AL., J APP POL SEI, vol. 88, no. 2, pages 369 - 379
GANAPATHY R ET AL: "IMMOBILIZATION OF ALPHA-CHYMOTRYPSIN ON OXYGEN-RF-PLASMA FUNCTIONALIZED PET AND PP SURFACES", JOURNAL OF BIOMATERIALS SCIENCE. POLYMER EDITION, VSP, UTRECHT, NL, vol. 9, no. 4, 1 January 1998 (1998-01-01), pages 389 - 404, XP002928810, ISSN: 0920-5063 *
NOSWORTHY, N.J. ET AL.: "A New Surface for Immobilizing and Maintaining the Function of Enzymes in a Freeze-Dried State", BIOMACROMOLECULES, vol. 10, 2009, pages 2577 - 2583
OGINO A ET AL: "Protein grafting onto chitosan surafce using low temperature microwave plasma treatment", PLASMA SCIENCE, 2010 ABSTRACTS IEEE INTERNATIONAL CONFERENCE ON, IEEE, PISCATAWAY, NJ, USA, 20 June 2010 (2010-06-20), pages 1, XP031721882, ISBN: 978-1-4244-5474-7 *
YIN ET AL., PPP, vol. 6, no. 1, pages 68 - 75
YIN, YONGBAI ET AL.: "Plasma Polymer Surface Compatible with a CMOS Process for Direct Covalent Enzyme Immobilization", PLASM. PROCESS. POLYM., vol. 6, 2009, pages 68 - 75

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