DE102010027936A1 - Immobilization of enzyme using plasmas - Google Patents

Abstract

The invention relates to an enzyme carrier system, obtainable by treating plastic surfaces with cold plasmas (low-temperature plasmas) and then immobilizing enzymes on the treated surfaces to increase the enzyme activity and the stability of use of immobilized enzymes. The invention is characterized in that the plasma treatment takes place without spacer-forming nitrogen-containing compounds with carbon chains.

Description

The invention relates to an enzyme-carrier system obtainable by 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 application stability of immobilized enzymes.

State of the art

Enzymes have hitherto been bound to plastic surfaces by a combination of physical and chemical techniques. 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 happens after Bosley et al. JAOCS 74_ 107-111 (1997) often by wetting with a water-soluble alcohol as a wetting agent. Another approach is the treatment with physical plasmas using spacer-forming nitrogen-containing compounds having carbon chains, such as e.g. B. Amines to the plasma gases. The aim is the formation of amino-functionalized surfaces 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. J App Pol Sci Volume 88 Issue 2 p 369-379 ). Frequently, additional spacer molecules such. As glutaraldehyde or the like as polymers or polymer combinations z. B. a polymer of α-hydroxyalkyleneamine ( US4757014 ) used. 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 the US Patent 5344701 is used as a coupling agent azalactone or azalactone polymer combinations using high energy radiation to generate radicals at the surface. in the WO2007 / 000163 Disulfite functions are used to couple polypeptides to carriers using radiation. Fields of application of these systems are molecular biology, biochemistry, pharmacology and medical diagnostics. In the patent DE 69737654T2 the use of plasma radiation for crosslinking is included, ethylene glycol bis (succinimidyl succinate) preferably being used here as an additional crosslinking agent.

Disadvantage of the prior art

The disadvantage consists partly in the complex plasma processes used and in the likewise 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. For a wide industrial use z. As in the chemical industry, the use of such elaborately prepared enzyme-carrier systems is usually not given.

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 being carried out without spacer-forming, nitrogen-containing compounds with carbon chains. The term "spacer-forming, nitrogen-containing organic compounds with carbon chains" are understood in accordance with the teachings of this invention, chemical compounds containing a carbon-nitrogen compound, such as. As amines or amides, with the aim of forming nitrogen-functionalized surfaces.

These low-temperature plasmas are discharges in gases, which are generated by means of high-energy radio-frequency technology in the range of low-frequency to high-frequency waves up to microwaves.

These plasmas can be used both in vacuum reactors in the low pressure range and in the normal pressure range, eg. B. with jet, barrier and hollow cathode discharges are operated. 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, more preferably esterases or lipases and the class of oxygenases, preferably oxidoreductases, more preferably monooxygenases, especially preferably Baeyer-Villiger monooxygenases, optimally modified. By suitable selection of plasma treatment methods, a multiple increase in enzyme activity over untreated comparable plastic carriers is achieved during immobilization. The increase of 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 is against this classic wet chemical methods the use of z. T. 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 substrates, preferably polypropylene plastics have been 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 oxy gases can be circumvented by the suitable choice of a carrier, preferably a polyalkene carrier such as 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. This may contain nitrogen-containing gases such. B. ammonia are admixed. As plasma sources, both microwave plasmas and radio-frequency plasmas are 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 by the straightforward, simple and cost-effective route with good up-scaling capabilities have been studied for 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.

• – Steigerung der Enzymaktivität und der Stabilität von Esterasen durch Vorbehandlung der Trägeroberfläche mit einem Gasentladungsplasma zur Immobilisierung
• – Steigerung der Enzymaktivität und der Stabilität von Lipasen durch Vorbehandlung der Trägeroberfläche mit einem Gasentladungsplasma zur Immobilisierung
• – Steigerung der Enzymaktivität und der Stabilität von Oxygenasen durch Vorbehandlung der Trägeroberfläche mit einem Gasentladungsplasma zur Immobilisierung

The advantages in detail are:

• - Increasing the enzyme activity and the stability of esterases by pretreating the support surface with a gas discharge plasma for immobilization
• - Increasing the enzyme activity and the stability of lipases by pretreatment of the support surface with a gas discharge plasma for immobilization
• - Increasing the enzyme activity and the stability of oxygenases by pretreatment of the carrier surface with a gas discharge plasma for immobilization

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 other plastics without the addition of auxiliary components in the plasma gas used, which should serve in the 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 marked increase in the enzyme activity of the enzyme-carrier systems is achieved when using the plasma-treated plastic carriers in comparison to the untreated carriers. Furthermore, an improvement of the storage and use stability is achieved.

The invention is explained in more detail below with reference to embodiments, without limiting the invention to these examples.

embodiments

Embodiment 1

Plasma treatment in 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.

Enzyme immobilization using the example of CalB lipase:

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 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 activity is determined by 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 μmol of 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 10 s effective plasma treatment time in the pulsed plasma with a pulse rate of 10/90 on / out and a pulse rate of 10 Hz 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 immobilized PestE esterase

The activity is determined 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 μl of 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.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 4757014 [0002]
• US 5344701 [0002]
• WO 2007/000163 [0002]
• DE 69737654 T2 [0002]

Cited non-patent literature

• Bosley et al. JAOCS 74_ 107-111 (1997) [0002]
• Yin et al PPP Volume 6 Issue 1 p68-75 [0002]
• Alvarez et al. J App Pol Sci Volume 88 Issue 2 p 369-379 [0002]

Claims (10)

Enzyme-carrier system obtainable by treatment of plastic surfaces with cold plasmas (low-temperature plasmas) and subsequent immobilization of enzymes on the treated surfaces, characterized in that the plasma treatment takes place without spacer-forming, nitrogen-containing compounds having carbon chains. Enzyme-carrier system according to claim 1, characterized in that act as plastic surfaces polyalkene surfaces, preferably polypropylene surfaces. Enzyme-carrier system according to claim 1 or 2, characterized in that argon or an argon / oxygen mixture or an argon / air mixture is used as the plasma gas. Enzyme-carrier system according to claim 3, characterized in that the plasma gas additionally contains nitrogen and / or ammonia. Enzyme-carrier system according to one of claims 1 to 4, characterized in that the enzymes are enzymes of the class of 5.1. Hydrolases 5.1.1. preferably carboxylesterases, 5.1.2. particularly preferred esterases or lipases or 5.2. oxygenases 5.2.1. preferably oxidoreductases, 5.2.2. particularly preferred monooxygenases, 5.2.3. especially preferred Baeyer-Villiger monooxygenases, is. Enzyme-carrier system according to one of claims 1 to 5, characterized in that the treated plastic surface has a hydrophilization of the support surface of less than 30 degrees for the water contact angle, preferably less than 10 degrees. Enzyme-carrier system according to one of claims 1 to 6, characterized in that the carriers are in the form of plates, nets, membranes or powders. Enzyme-carrier system according to claim 7, characterized in that the carriers are porous or non-porous. A process for the preparation of enzyme-carrier systems according to any one of claims 1 to 8, 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, takes place. Process for the preparation of enzyme-carrier systems according to Claim 9, characterized in that microwave plasmas or radio-frequency plasmas are used in the low-pressure or atmospheric pressure range.