WO2011131594A1

WO2011131594A1 - Immobilizing enzymes using plasmas

Info

Publication number: WO2011131594A1
Application number: PCT/EP2011/056069
Authority: WO
Inventors: 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: 2010-04-19
Filing date: 2011-04-16
Publication date: 2011-10-27
Also published as: DE102010027936A1

Abstract

The invention relates to a method for treating plastic surfaces using cold plasmas (low-temperature plasmas) and subsequently immobilizing enzymes on the treated surfaces, characterized in that the plasma treatment leads to highly activating the plastic surface at a maximum depth of 3 nm and the plasma treatment takes place without spacer-forming compounds comprising nitrogen and having carbon chains, wherein microwave plasmas in the GHz range or radio frequency plasmas (1 kHz - 100 MHz) in the vacuum pressure or atmospheric pressure range are used for highly activating the plastic surface.

Description

Immobilization of enzymes using plasmas Description

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.

State of the art

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. The goal is the formation of surfaces functionalized with amino groups for the subsequent covalent attachment of enzymes in a second, subsequent step (Yin et al., PPP Volume 6 Issue 1 p68-75, Alvarez et al.) App Pol Volume 88 Issue 2 p 369- 379). Frequently, additional spacer molecules, such as, for example, glutaraldehyde or the like, such as polymers or polymer combinations, for example a polymer of α-hydroxyalkyleneamine (US Pat. No. 4,757,014), are used for this second subsequent step. In US Pat. No. 4,757,014, 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. In US Pat. No. 5,344,701 azalactone or azalactone polymer combinations are used as the coupling agent using high energy radiation to generate surface radicals. In WO2007 / 000163 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. in the 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.

[0003] 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. Such 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.

[0004] The subject of the publication by Nosworthy, NJ, et al., "A New Surface for Immobilizing and Maintaining the Function of Enzymes in a Freeze-Dried State", Biomacromolecules, 2009, 10, 2577-2583, is the immobilization of proteins such as HRP and catalase on pill (plasma ion immersion implantation) -treated polyethylene. The activation takes place only after the Plll process in contact with the atmospheric oxygen - Abreaktion of the radicals to oxygen-functional groups - instead.

Disadvantage of the prior art

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. However, 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. However, for a broad industrial use, for example in the chemical industry, the use of such elaborately prepared enzyme-carrier systems is generally not given. The disadvantage of the publication of YIN et al. This is because the CMOS coatings require annealing processes that are performed at 350-400 ° C.

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. In addition, in our experience 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).

Object of the invention

The invention had the object to eliminate the disadvantages of the technical solutions described in the prior art.

Solution of the task

The problem has been solved according to the features of the claims.

According to the invention, the preferably used commercially available plastic carrier materials, preferably polypropylene 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. By the term "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. In 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.

These 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. Preferably, 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. Thus, 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. By suitable selection of plasma treatment methods, a multiple increase in enzyme activity over untreated comparable plastic carriers is achieved during immobilization. The increase in the number of interaction sites for the enzymes on the surface of the plastic carriers are made possible by the plasma treatment without the use of additional so-called wet-chemical processes such as the deposition of mediator layers from solutions. It eliminates the use of z.T. of these classic wet chemical methods. toxic solvents and their disposal after use and consuming cleaning and processing steps such as rinsing and time-consuming drying of the treated plastic materials.

Surprisingly, it has been found that the enzyme activity of immobilized enzymes, in particular the class of esterases, lipases and oxygenases on plastic supports, preferably polypropylene plastics were improved with the aim of industrial use of these immobilized catalyst systems. The invention enables an improvement in the stability of the enzyme catalyst systems and thus the multiple use of these catalyst systems with almost constant high activity. Thus, a significant cost reduction in industrial use based on the production turnover over conventional enzyme catalysts is made possible.

Detailed description of the invention Surprisingly, according to the invention, 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. As plasma sources both microwave plasmas and radio frequency plasmas are each suitable in the low pressure range as well as under atmospheric pressure. 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.

Advantage of the invention over the prior art

The materials obtained in a straightforward, simple and cost-effective way with a good possibility of up-scaling have been subjected to studies on long-term stability under different storage conditions and over several cycles of biocatalysis. The materials showed good stability. Thus, very inexpensive and stable enzyme-carrier systems are obtained in relation to the previously known immobilization strategies by means of preparation of the surface by plasma processes. These can be prepared according to the invention to industrial scale.

According to the invention no covalent attachment of enzymes is used, but a chemisorption, which does not take place on plasma-polymerized materials, but on plastic surfaces that are chemically highly activated with special plasmas. This means that the plasma processes are optimized in order to achieve high densities of reactive chemical groups such as alcohols, ethers, carboxylic acids and peroxides in a suitable mixture (polar and nonpolar groups). Thereafter, the immobilization of enzymes takes place. Plasma high activation is not a pure gas phase process polymerizable gases, that is not associated with the supply of reactive gases such as acetylene, which leads to a layer formation described in the prior art. 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.

Surprisingly, it has been found that 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.

The advantages in detail are:

Increasing the enzyme activity and the stability of esterases by pretreatment of

Carrier surface with a gas discharge plasma for immobilization

Increasing the enzyme activity and the stability of lipases by pretreatment of the

Carrier surface with a gas discharge plasma for immobilization

Increasing the enzyme activity and the stability of oxygenases by pretreating the carrier surface with a gas discharge plasma for immobilization

Annealing processes, which are carried out at 350-400 ° C, are among the

Plasma uptake activations according to the invention are not necessary

It has also been possible to show that enzyme immobilization is dependent both on the type of enzyme and on the nature of the carrier and the type of surface pretreatment.

For the first time with the present invention, 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. In this case, 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.

According to the invention 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 invention will be explained in more detail with reference to embodiments, without limiting the invention to these examples.

embodiments

Exemplary Embodiment 1 Plasma Treatment in the RF Plasma

The polypropylene powder is treated in an RF plasma system at 27.2 MHz. For this purpose, according to the embodiment, 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. For this purpose, according to the embodiment, 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.

Activity determination of immobilized CalB lipase

The determination of the activity by means of the pH-stat method. These automatic Titrationsanlage (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 μιηοΐ butyric acid per minute by the immobilized enzyme.

Embodiment 2

Plasma treatment in microwave plasma:

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. For this purpose, 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. For this purpose, 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.

Enzyme immobilization using the example of PestE esterase:

500 mg of the plasma-treated polypropylene powder is incubated in a 15 ml glass vessel 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 immobilization buffer and dried overnight in vacuo. Storage before the activity measurements takes place at 4 ° C.

Activity determination of the immobilized PestE esterase

The determination of the activity is carried out by means of a rapid test in 24-well microtiter plates. To this is added 400 μl 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 μΐ 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.

Claims

claims

1. A process for the treatment of plastic surfaces with cold plasmas (low temperature plasmas) and subsequent immobilization of enzymes on the treated surfaces, wherein the plasma treatment leads to a high activation of the plastic surface with maximum depth of 3 nm and the plasma treatment without spacerbildende, nitrogen-containing compounds with carbon chains takes place in that microwave plasmas in the GHz range or radio frequency plasmas (1 kHz - 100 MHz) in the low-pressure or atmospheric-pressure range are used for highly activating the plastic surface.

2. The method according to claim 1, characterized in that microwave plasmas in the range of 100 MHz to 10 GHz, preferably 2.54 GHz, are used.

3. The method according to claim 1 or 2, characterized in that RF plasmas are ignited with powers of 1 W to 100 W or microwave plasmas with 100 W to 1500 W at gas flow rates of 1 to 100 sccm.

4. The method according to any one of claims 1 to 3, characterized in that the plastic surface is treated for 1 ms to 600 s, preferably 10 ms to 1 s, more preferably 10ms to 100ms long.

5. The method according to any one of claims 1 to 4, characterized in that reactive species such as radicals and ions are generated for the high activation of the surface, wherein the ions are accelerated with voltages of at most 500 V.

6. The method according to any one of claims 1 to 5, characterized in that act as plastic surfaces polyalkene surfaces, preferably polypropylene surfaces.

7. The method according to any one of claims 1 to 6, characterized in that is used as the plasma gas argon or an argon / oxygen mixture or an argon / air mixture.

8. The method according to claim 7, characterized in that the plasma gas additionally contains nitrogen and / or ammonia.

9. The method according to any one of claims 1 to 8, characterized in that the enzymes are enzymes of the class of

9.1. Hydro read,

9.1.1. preferably carboxylesterases,

9.1.2. particularly preferred esterases or lipases or

9.2. oxygenases

9.2.1. preferably oxidoreductases,

9.2.2. particularly preferred monooxygenases,

9.2.3. especially preferred Baeyer-Villiger monooxygenases,

is.

10. The method according to any one of claims 1 to 9, characterized in that after the plasma treatment, the immobilization of the enzymes from solutions with and without buffer by storing in an enzyme solution for at least 1 min to several hours or days, preferably 8-16 Hours with or without stirring.

11. Enzyme-carrier system with a plastic surface as a carrier which has been highly activated with maximum depth of 3 nm and a hydrophilization of less than 30 degrees for the water contact angle, preferably less than 10 degrees, obtainable by a method according to one of claims 1 to 10 ..

12. Enzyme carrier system according to claim 11, characterized in that the enzymes are enzymes of the class of

12.1. Hydrolases

12.1.1. preferably carboxylesterases,

12.1.2. particularly preferred esterases or lipases or

12.2. oxygenases

12.2.1. preferably oxidoreductases,

12.2.2. particularly preferred monooxygenases,

12.2.3. especially preferred Baeyer-Villiger monooxygenases,

is.

13. Enzyme carrier system according to claim 11 or 12, characterized in that the carriers are in the form of plates, nets, membranes or powders.

14. Enzyme carrier system according to claim 13, characterized in that the carriers are porous or non-porous.