Motivation Das große Potential von Enzymen als stereoselektive Katalysatoren in der asymmetrischen Wirkstoffsynthese wird immer häufiger ausgenutzt. Der technologische und synthetische nutzen dieser Biokatalysatoren kann deutlich erweitert werden, wenn sie in organischen Lösungsmitteln anstelle ihres natürlichen, wässrigen Reaktionsmedium eingesetzt werden. Da aber Enzyme in fast allen organischen Lösungsmitteln unlöslich sind, sind dort ihre katalytischen Aktivitäten gering. Die Verbesserung der enzymatischen Aktivität ist von großem wissenschaftlichen und wirtschaftlichen Interesse. In Vorarbeiten haben wir eine neuartige Aktivierungs-Methode entwickelt, die auf der Trägerung von Enzymen in Nanophasen-separierte amphiphilen Polymernetzwerken beruht.
- Synthese von amphiphilen Netzwerken
- Analyse und Charakterisierung der chemischen Eigenschaften und Netzwerkmorphologie in Bezug auf den Einfluss auf die Enzymaktivität
- Ausrichtung der Enzyme in amphiphilen Netzwerken zur Erhöhung der Enzymaktivität
- Synthese von amphiphilen Mikropartikeln für den Aufbau eines Säulenreaktors
- Synthese von amphiphilen Membranen zur Proteintrennung
- Synthese von chiralen amphiphilen Netzwerken zur Verbesserung der Enantioselektivität von Enzymen
Biocatalytically active Nanofibers for Organic Solvents
Entrapment of Enzymes in Electrospun Polymer Nanofibers for the a Biocatalytic StirrerRamona Plothe, Ina Sittko, Joerg C.TillerBiocatalysis has become an alternative to classical organo-metal catalysis, but is often limited to aqueous media, because most enzymes show very low activities in organic solvents. A common tool to active enzymes in organic media is to immobilize them on suited carriers, but due to diffusion limitations this does not results in the highest possible activities. We have overcome this problem by electrospinning enzymes from aqueous solutions containing the polymer poly(2-ethyloxazoline), which results in highly biocatalytically active nanofibers.
The use of nanofibers for enzyme entrapment is a field of study with enormous potential. In the present work, poly(2-ethyloxazoline) (PEtOx) was used as polymer component for direct electrospinning of different enzymes in aqueous media (Figure 1). The process was optimized by varying process parameter to obtain nanofibers with few hundreds of nanometers in diameter that contain up to 10 wt% protein. It was found that not only the shape and quality of the fibers but also the activity of the enzymes in organic solvents is greatly influenced by the polymer content in the spinning solution.
Figure 1: Electrospinning process, resulting nanofibers and carrier activities of PEtOx NF with 5wt% CaLB and Novozym® 435 in toluene.
A series of enzymes, including laccase, horseradish peroxidase, alcohol dehydrogenase, chymotrypsin, and several lipases was prepared under optimized conditions. All enzymes safe alcohol dehydrogenase survived the process with only slightly diminished activity. Measuring the activity of the enzyme loaded fibers in n-heptane revealed that the biocatalysts are activated by up to two orders of magnitude in all cases compared to the respective enzyme powder suspended in the same solvent. The highest activation was found for proteases, which were also found to be the most stable in the spinning process. The electrospun nanofibers loaded with lipase B from Candida antarctica (CaLB-NF) were applied as biocatalyst systems for the esterification of 1-octanol and lauric acid in n-heptane and toluene, respectively. Thereby the enzyme activity depends on the diameter of thefibers and water content of the reaction media, e.g. adding of 1 vol% water to the reaction mixture in n-heptane increasesthe carrier activity of CaLB-NF by more than 100%. However, in contrast to literature, the highest enzyme activity wasfound toluene, which is due to a minor swellablity of the matrixpolymer PEtOx in this solvent. Using electrospunCaLB under optimized conditions reveals a higher carrier activity than the commercial immobilized CaLB Novozym® 435 with ten times less immobilized protein (Figure 1).
Figure 2: Biocatalytically active stirrer.
Charge A and B describe two different lipaseassay solutions in n-heptane with the same concentration of educts where the stirrer is dipped into alternately. The reaction was followed by determining the CaLB catalyzed ester formation of 1-octanol and lauric acid. The fibers were electrospun from an aqueous solution that contains 20 wt% PEtOx and 0.8 wt% CaLB, the enzyme content in the resulting fiber was 4 wt%.
Another advantage of using electrospinning is the opportunity of easily modifying various surfaces. The electrospinning of PEtOx/CaLB fibers onto a stirrer is used to realize a biocatalytic stirrer for organic solvents (Figure 2). As seen in figure 2, the fibers are highly stable on the stirrer and could repeatedly switched between two reactors without losing activity. R. Plothe, I. Sittko, F. Lanfer, M. Fortmann, M. Roth, V. Kolbach, J. C. Tiller
Poly(2-ethyloxazoline) as matrix for highly active electrospun enzymes in organic solvents
Biotechnology and Bioengineering 114 (1), 39-45 (2017)
Organosoluble Artificial Metalloenzymes
Melanie Leurs, Stefan Konieczny and Jörg C. Tiller
Polymer enzyme conjugates as chiral ligands for sharpless dihydroxylation of alkenes in organic solvents
Enzymes are becoming a serious alternative to organometallic catalysts in the synthesis of fine chemicals. There is a great variety of chemical reactions that can be catalyzed by enzymes, particularly when working in organic solvents. However, many reactions cannot be catalyzed by enzymes. In order to enhance the reaction spectrum of enzymes in organic solvents, we modified the active center of organosoluble poly(2-methyloxazoline)-enzyme-conjugates with osmate to create artificial enzymes that are capable of catalyzing the dihydroxylation of alkenes with high enantioselective control in organic solvents. These catalysts are the first examples of organosoluble artificial metalloenzymes.
Artificial metalloenzymes (AMEs) have an emerging field in catalysis. So far no organosoluble examples are known, which greatly restricts the use of such systems. In general, AMEs are merging molecular recognition ability of proteins and broad reactivity scope of organometallic catalysts to form superior hybrid catalysts. We chose the osmate-catalyzed Sharpless dihydroxylation of alkenes as model reaction for our investigations (Figure 1). Conjugates of enzymes and poly(2-methyl-oxazoline) (PMOx), generated by coupling of the terminal amino group of the polymer and the amino groups of the enzyme via pyromellitic acid dianhydride, are soluble in organic media, e.g. chloroform.
Figure 1: Sharpless dihydroxylation with polymer enzyme conjugates as ligand.
One great advantage of these organo-soluble PECs is that they can act as amphiphilic core-shell nanocontainers. Potassium osmate is not soluble in chloroform. After adding the salt to a PEC solution in this medium, it can only be dissolved by being complexed to a hydrophilic core, which is the protein part of the PEC. Thus, the osmate is forced into the chiral environment of the enzyme and thereby incorporated by dative anchoring. Investigations of a number of different conjugates of PMOx-enzyme conjugates revealed that the enantioselctive control of the Sharpless dihydroxylation can be controlled by the nature of the enzyme even favoring S- and R-configuration. The greatest selectivity was found for the laccase-PEC. Optimizing the reaction conditions with laccase (from Trametes vesicolor) PECs strongly indicates that osmate is located in the active site of this enzyme by a copper to osmate exchange (Figure 2). Therefore copper was previously deliberately removed from laccase by dialysis of the PECs against an aqueous ethylenediaminetetraacetic acid (EDTA) solution.
Figure 2: Scheme of the suggested copper type 1 to osmate exchange by treatment of the laccase-PEC with EDTA as Cu-scavenger.
It was shown that the resulting artificial metalloenzyme catalyzes the dihydroxylation of alkenes in chloroform and affords highly enantioselective product formation. Thereby product enantioselectivities up to 99.4 % ee were achieved, which even exceeds the classical Sharpless catalysts. Thus, we could transfer the concept of artificial metalloenzymes from water to organic solvents with even higher selectivity. Current work is dedicated to broaden the concept to other enyzmes and further reactions.
S. Konieczny, M. Leurs, J. C. Tiller
Polymer Enzyme Conjugates as Chiral Ligands for Sharpless Dihydroxylation of Alkenes in Organic Solvents
Defined Polymer Nanostructures for High Enzyme Activity
Revealing the origin of enzyme activation in APCNs in organic solvents
Ina Sittko and Jörg C. Tiller
Enzymes are natural catalysts with exceptionally high activity and selectivity in water. Although, they selectively catalyze reactions in organic solvents as well, their activity is several orders of magnitude lower in such media. Since most fine chemicals are not soluble in water, high enzyme activity is required for the practical use of biocatalysts in industrial syntheses. We have previously discovered that amphiphilic polymer conetworks (APCNs) activate entrapped enzyme in organic media. In this study, we have developed APCNs tailored to clarify the working mechanism of the biocatalytic materials. It was found that the defined nanostructure of APCNs is indeed the major factor for activation of entrapped enzymes.
The general concept of activation of enzymes in APCN is that the enzyme is located in the hydrophilic nanophase, while the substrate diffuses to the hydrophobic nanophase, is converted at the large interface under enzyme catalysis and the product diffuses out of the conetwork via the same nanophase. According to this concept, the best activity should be obtained in the APCN with two interconnected phases, which is typical for APCNs with some 50 % of both different polymer volume fractions. The enzyme activation should be solvent independent, if the activation is achieved by the large interface. So far, this has never been observed for all explored biocatalytic APCNs, most likely, because there are influencing parameter, such as swelling, that over-lap the true activation effect.
Figure 1. Sketch of the synthesis of APCNs with an entrapped enzyme.
In this study, we prepared two new APCNs, which allow entrapment of enzymes during preparation by copoly-merization of n-butyl acrylate and 2-ethylhexyl acrylate, respectively, with telechelic methacrylamide terminated poly(2-methyloxazoline) (PMOx). Both APCNs have a distinguished nanostructure shown by atomic force microscopy (see Fig. 2 on the example of PBuAc-l-PMOx). Their major advantage is that PBuAc-l-PMOx swells in toluene but not in n-heptane, while the structurally close PEhAc-l-PMOx is swellable in both solvents. This gives us the opportunity to distinguish between swelling and structural effects on enzyme activity. The common lipase Cal B was entrapped in a series of the APCNs.
Figure 2. Left (a): Atomic force microscopy image of a PBuAc-l-PMOx APCN with approximately 50 vol% of each polymer phase, measured in phase modus. Right (b): Specific activity of entrapped lipase in PBuAc-l-PMOx of different compositions in toluene.
Lipase Cal B entrapped in the conetworks is most active at a composition that contains some 50 wt.% PMOx in all cases. Further, the maximal activation of Cal B with respect to the suspended powder is some 20-fold independent on the solvent as long as the APCN is swellable. This supports the proposed mechanism of enzyme activation in APCNs.
Although the activation of some 20-fold within the APCNs is exceeded by other supporting materials, the formation of particles out of the bioactive amphiphilic conetworks has been found to overcome diffusion limitation and greatly activates the entrapped enzymes compared to the here presented polymer films. Thus, we will strive to prepare the here developed APCN as particles in future work.
I. Sittko, K. Kremser, M. Roth, S. Kuehne, S. Stuhr, J. C. Tiller
Amphiphilic Polymer Conetworks With Defined Nanostructure and Tailored Swelling Behavior for Exploring the Activation of an Entrapped Lipase in Organic Solvents
Polymer 64, 122-129 (2015)
Biocatalyzed reactions in APCN particles in organic solvents
Investigations on diffusion limitations
Ina Schönfeld, Stephan Dech, Benjamin Ryabenky, Bastian Daniel, Britta Glowacki, Reinhild Ladisch and Jörg C. Tiller
The use of enzymes as biocatalysts in organic media is an important issue in modern white biotechnology. However, their low activity and stability in those media often limits their full-scale application. Amphiphilic polymer conetworks (APCNs) have been shown to greatly activate entrapped enzymes in organic solvents. Two different APCNs as nanostructured microparticles showed greatly increased activities of entrapped enzymes compared to those of the already activating membranes.
In the past, we have shown that amphiphilic polymer conetworks are excellent activating carriers for biocatalysts in organic solvents. Although great activations could be achieved, the reactions were generally diffusion controlled and thus the full potential of these networks was not reached. We investigated potential increase of the activity of different enzymes in different APCNs by preparing microsized particles from these materials. First, we demonstrated this on the example of APCN particles based on PHEA-l-PDMS loaded with α-Chymotrypsin, which resulted in an up to 28000fold higher activity of the enzyme compared to the enzyme powder.
Suspension polymerization and aerosol polymerization were used to synthesize PHEA-l-PDMS microparticles of different compositions in a diameter range of 5 – 80 µm (Figure 1).
Figure 1: SEM images of PHEA-l-PDMS particles a) prepared by vigorously stirred emulsion polymerization, b) prepared by vigorously stirred emulsion polymerization and subsequent filtration through a 20 µm metal mesh and c) prepared by aerosol polymerization.
The activity of supported α-Chymotrypsin in these microparticles in n-heptane was strongly increasing with decreasing particle diameter. The highest specific activity was 56 U/g for particles with a diameter of 5 µm.
Another particle system consisting of PHEA-l-PEtOx (30/70) in a diameter range of 10 to above 50 µm (Figure 2), loaded with lipase from Rhizomucor miehei (RmL) was tested in different organic solvents. In all solvents, smaller particles showed ten to 100fold higher specific activities compared to the native enzyme. This was even true for the production relevant reaction mixture without additional solvent. In the latter case the RmL-loaded PHEA-l-PEtOx microparticles show nearly tenfold carrier activity with some 25-50fold lower enzyme content compared to a commercial product.
Figure 2: Specific activities (U/g enzyme) of RmL immobilized into PHEA-l-PEtOx (30/70) conetwork particles (RmL content 0.2 wt.%) in correlation to the average particles size (volumetric average). Assay was performed in chloroform.
Even the smallest particles showed a diffusion limitation of the reaction possibly by surface limited mass transport, which indicates that the here presented improvement of activity by particularization is still not the highest achievable activation of the APCN supported enzymes. Future investigations are directed towards the application of techniques to further increase the surface/bulk ratio and thus the activity of the biocatalytic APCNs.
I. Schoenfeld, S. Dech, B. Ryabenky, B. Daniel, B. Glowacki, R. Ladisch, J.C. Tiller
Investigations on Diffusion Limitations of Biocatalyzed Reactions in Amphiphilic Polymer Conetworks in Organic Solvents
Biotechnology and Bioengineering, in print (2013)
Polymer Enzyme conjugates with Poly(oxazoline)s
Organosoluble enzyme conjugates for biocatalysis
Stefan Konieczny and Jörg C. Tiller
Enzymes can be used as selective and highly active biocatalysts for the synthesis of complex organic compounds. By the usage of enzymes in organic solvents, their potential can be greatly extended and this is therefore in focus of current research. Here we report the synthesis of fully organosoluble poly(2-oxazoline) (POx) enzyme conjugates with different enzymes using a new coupling technique.
The cross metathesis of methyl undec-10-enoate 1 with dimethyl maleate 2 results in the products dimethyl dodec-2-enedioate 3 and methyl acrylate 4 (Fig.1 One way for the application of enzymes in organic media is the chemical modification with polymers. Through such a modification, the enzymes become soluble in the organic solvent and this leads to a higher catalytic activity.
The POXylation was carried out reacting pyromellitic acid dianhydride (PADA) as bifunctional reagent subsequently with ethylenediamine terminated POx and then with the NH2-groups of the respective enzymes.
It could be shown, that the PADA can be used to modify the polymers and use the derivatives without further modification.
Upon conjugation with the polymers, RNase A and lysozyme became fully soluble in DMF (1.4 mg/ml). These are the first examples of fully POXylated proteins, which become organosoluble (Figure 1).
Figure 1: ~7 mg lysozyme in 5 ml DMF: (a) native (b) modified with PADA (5.0 mg) only (c) modified with poly(2-methyl-1,3-oxazoline) (81.3 mg) and PADA (5.0 mg).
The synthesized enzyme conjugates were characterized by SDS-PAGE, isoelectric focusing, dynamic light scattering and size exclusion chromatography, which all indicated the full POXylation of the enzymes (Figure 2).
Interestingly, the degree of modification as well as the nature of the modified proteins is controlling the formation of aggregates in solution.
Figure 2: SDS-PAGE of lysozyme and the corresponding POx enzyme conjugates modified in DMF: (a) M Marker; Ly, lysozyme; A, lysozyme modified with poly(2-methyl-1,3-oxazoline); (b) B, lysozyme modified with poly(2-ethyl-1,3-oxazoline); (c) D, LyCon5
The modified lysozyme and a-chymotrypsin even partly retained their activity in water and in the case of lipases also in chloroform.
It could be demonstrated with a-chymotrypsin as example, that the molecular weight of the attached polymer significantly influences the activity in water depending on the size of the substrate.
In current and future experiments the solubility and enzymatic activity of the conjugates in other organic solvents will be tested.
By the usage of other POx and/or altering the modification parameters it might be possible to achieve solubility in other organic solvents as well. The developed strategy for the synthesis of polymer enzyme conjugates can also be used for further enzymes.
S. Konieczny, C.P. Fik, N.J.H. Averesch, J.C. Tiller
Organosoluble Enzyme Conjugates with Poly(oxazoline)s via Pyromellitic Acid Dianhydride, Journal of Biotechnology 159, 195-203 (2012)
APCN for activating enzymes in organic solvents
Poly(2-oxazoline)-based APCN take up enzymes during preparation
Stephan Dech, Jörg C. Tiller
The high functionality of modern materials is often achieved by a controlled nanostructure. Amphiphilic polymer conetworks (APCN) show such a nanostructure, which can be used to greatly activate entrapped enzymes in organic solvents. Unfortunately, the loading capacity and enzyme size are limited to less than 1 wt% loading and small enzymes. Here, we present a new APCN class that can be prepared in aqueous solutions and is capable of entrapping enzymes of any size with up to 11 wt.% loading. It was shown on the example of lipase that the entrapped enzymes are even higher activated in organic solvents than that in conventional APCNs.
Amphiphilic polymer conetworks represent a class of nanomaterials with great application potential, because they can combine the properties of two chemically different, immiscible polymers in one macroscopically homogeneous nanophasic material. Both polymer nanophases can act independently from each other. This is due to the fact that the conetworks consist of two polymers one crosslinked by the other, i.e. they are segmented conetworks. The copolymerization of Poly(2-ethyloxazoline) (PEtOx) macromonomers with 2-hydroxyethylacrylate (HEA) results in optically clear, homogeneous conetwork membranes (Fig. 1). DSC, DMA, and AFM measurements revealed that the membranes consist of two partially mixed polymer nanophases, which showed characteristic properties of amphiphilic conetworks (Fig. 2).
Figure 1: Photos of PHEA-l-PEtOx4.8conetwork in dried and water swollen state.
Lipase rhizomucor miehei was successfully entrapped prior to the polymerization step for first time in those kinds of conetworks. The high activity of immobilized RmL in water indicates the efficiency of this mild method to entrap enzymes.
The activity results for such captured RmL in organic solvents showed the superior properties of these new PHEA-l-PEtOx conetworks. Compared to the literature known PHEA-l-PDMS systems, an up to 6-fold higher specific activity and an up to 8-fold higher conetwork activity were obtained.
Figure 2. AFM images of PHEA-l-PEtOx4.8 conetworks with 50 and 70 wt.% PEtOx4.8(from left to right). Images were recorded in tapping, phase mode. Phase contrast here is 8 ° (left) and 21° (right).
This makes PHEA-l-PEtOx conetworks outstanding materials for enzyme entrapment and their use as nanoreactors for biocatalytic reactions in organic media. Also numerous other larger functional structures such as nanoparticles, antibodies or even ion pumps might be entrapped into such conetworks.
S. Dech, V. Wruk, C.P. Fik, J.C. Tiller
Amphiphilic Polymer Conetworks Derived from Aqueous Solutions for Biocatalysis in Organic Solvents
Polymer 53 (3), 701-707 (2012)