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Biocatalytically active thin films via self-assembly of 2deoxy-D-ribose-5-phosphate aldolase/PNIPAm conjugates Shuhao Zhang, Carolin Bisterfeld, Julia Bramski, Nane Vanparijs, Bruno G. De Geest, Jörg Pietruszka, Alexander Böker, and Stefan Reinicke Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00645 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Bioconjugate Chemistry

BIOCATALYTICALLY ACTIVE THIN FILMS VIA SELFASSEMBLY OF 2-DEOXY-D-RIBOSE-5-PHOSPHATE ALDOLASE/PNIPAM CONJUGATES Shuhao Zhanga),e), Carolin Bisterfeld b), Julia Bramski b), Nane Vanparijsc), Bruno G. De Geestc), Jörg Pietruszka b),d), Alexander Böker a),e) and Stefan Reinicke a)

a) Department of Functional Protein Systems and Biotechnology, Fraunhofer Institute for Applied Polymer Research (IAP), Geiselbergstraße 69, 14476, Potsdam-Golm, Germany b) Institute of Bioorganic Chemistry, Heinrich Heine University of Düsseldorf at Forschungszentrum Jülich, Stetternicher Forst, 52426 Jülich, Germany c) Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium d) IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany e) Polymer Materials and Polymer Technologies, University of Potsdam, 14476, PotsdamGolm, Germany E-Mail: [email protected]

ABSTRACT 2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a biocatalyst that is capable of converting acetaldehyde and a second aldehyde as acceptor into enantiomerically pure mono- and diyhydroxyaldehydes, which are important structural motifs in a number of pharmaceutically active compounds. However, substrate as well as product inhibition requires more a sophisticated process design for the synthesis of these motifs. One way to do so is to couple aldehyde conversion with transport processes, which in turn would require an immobilization of the enzyme within a thin film that can be deposited on a membrane support. Consequently, we developed a fabrication process for such films, which is based on the formation of DERA/poly(N-isopropyl acrylamide) conjugates that are subsequently allowed to self-assemble

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at an air/water interface to yield the respective film. In this contribution, we discuss the conjugation conditions, investigate the interfacial properties of the conjugates and finally demonstrate a successful film formation under preservation of enzymatic activity.

INTRODUCTION Enzymes are versatile catalysts for both lab scale and industrial applications1, as they can be considered very effective, often showing a high chemo- and stereoselectivity. However, enzymes are often prone to structural instability outside their natural environment and isolating them from the reaction mixture upon product purification is often laborious. In that respect, immobilization of the enzyme can be of great benefit. For instance, enhanced stability occurring upon immobilization ensures a long term use and recyclability of the enzyme.2-4 An additional advantage in some cases is the opportunity to establish an alternative process design such as a continuous operation mode.3 To date, many different strategies for immobilization of enzymes have been reported1, amongst which are ultrathin enzyme containing membranes on organic or inorganic supports.5-7 An ultrathin membrane, not thicker than a few nanometers is typically free of diffusion limitation and requires only a small amount of enzyme. Although these small amounts might also limit the overall catalytic activity of the membrane, a reasonable performance can still be achieved if the immobilization process is designed in a way that the specific activity of the immobilized enzyme is not or only slightly compromised. Different ways of producing ultrathin enzyme containing films are available, including the Langmuir-Blodgett or -Schaefer technique, other self-assembly techniques at interfaces and electropolymerization in the presence of enzymes on electrode surfaces.8 Although ultrathin enzyme containing films so far are mostly used in the biosensor field9, they have also great potential in biocatalysis.3 2-Deoxy-D-ribose-5-phosphate aldolase (DERA), is a particular representative of a wide class of enzymes that catalyze the C-C linking reaction between two organic carbonyl compounds.10-12 Because of its exclusive ability to utilize an aldehyde instead of another carbonyl function as the nucleophile, yielding another aldehyde as product which again can be subjected to an aldol reaction, DERA has been applied to synthesize a broad range of dihydroxy aldehydes with high yields and high stereoselectivity.13-15 These compounds are typically more difficult to obtain via conventional synthetic strategies. An example for the industrial application of DERA is its use in


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an economically viable process for the synthesis of a key building block for the pharmaceutical blockbuster atorvastatin.16 However, the main drawback of DERA being used in such a process is its sensitivity to industrially relevant concentrations of the substrate and the occurrence of product inhibition. Substrate sensitivity can be enhanced by genetic engineering17,

18

while

product inhibition can be avoided by coupling the catalytic transformation with transport processes. Here, immobilization of DERA in thin films on membrane supports becomes relevant. Next to the avoidance of product accumulation, the proposed thin film concept would also enable to limit the contact time between DERA and substrate by adjustment of film thickness and flow rate, thereby allowing the isolation of the intermediate product of the two-step aldol reaction, which is monohydroxyaldehydes. Structures of this type constitute relevant building blocks for pharmaceutical compounds as well.19 Immobilization of DERA has already been reported several times.20-23 However, in these contributions, nanomaterials in bulk had been used as supports, and no focus was put on the product accumulation issue or the opportunity for isolation of the intermediate products. Furthermore, the catalytic activity of the immobilized DERA was poor21,

22

or not even

20

reported. One way to create thin enzyme containing films is to bind the protein to a preformed polymer film. Such an approach has been followed already by our group.24 Other methods rely on the in situ entrapment of the enzyme during polymerization of respective monomers.25, 26 Another approach to create protein-containing films as thin as monolayers, which has been also frequently used by us, is to first equip the enzyme with polymer chains followed by selfassembly of the formed conjugates at an interface.27-30 The polymer chains form the film matrix after the self-assembly step but can also enhance the surface activity of the enzyme which is beneficial for the self-assembly process.29,

30

While the first two methods described do not

always lead to films with an even distribution of protein over the whole film area or are limited in their minimum achievable thickness, the latter method ensures these two features, which compensates for the synthetic effort prior to film formation. Enzyme-polymer conjugates are accessible via three main routes.31 The first route is the grafting-from approach.32, 33 Here, a functional group that is able to mediate or initiate a polymerization process is introduced to an amino acid side residue of the protein. The second route, grafting-through, is based on the attachment of polymerizable groups to the protein surface transferring the protein into a macromonomer which can be incorporated into a growing polymer chain upon a polymerization



process.34 The third method, grafting-to, relies on the attachment of preformed polymers to the protein.35 This approach is mediated either through covalent attachment of a reactive functional group of the polymer to a corresponding amino acid side-chain, or by a ligand-apoprotein interaction. The grafting-to approach has the advantage of a better control over the polymerization process including the opportunity of a thorough characterization of the polymer prior to the conjugation step, compared to the other grafting techniques. In this contribution we present the synthesis and the self-assembly of DERA/poly(Nisopropylacrylamide) (PNIPAm) conjugates via the grafting-to approach (Scheme 1) and prove that enzymatic activity can be maintained during all manipulation steps, which is a necessary pre-requisite for the successful generation of a biocatalytically active film. As PNIPAm is soluble in protein friendly, aqueous media, it is in principle suitable for being used in the conjugation step. Additionally, it is a temperature-responsive polymer, showing a volume phase transition above the lower critical solution temperature (LCST). Utilizing this transition can be used later on to tune flux rates and catalytic activity on demand in the final biocatalytically active film.36, 37

Scheme 1. Formation of a catalytically active DERA containing thin film by self-assembly of respective DERA/PNIPAm conjugates.


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RESULTS AND DISCUSSION RAFT Polymerization of NIPAm with PDS-CTA The polymers for the conjugation were synthesized via RAFT polymerization. For that, a chain transfer agent with a cysteine-reactive pyridyl disulfide group (PDS-CTA) was used. The polymerization process is shown in Scheme S-1. To investigate the influence of the polymer chain length on the conjugation efficiency of the polymers, as well as the activity and the acetaldehyde tolerance of the resulting conjugate, polymers with different targeted degrees of polymerization (DP) were synthesized, in particular 50, 100 and 500. The resulting polymers were analyzed by 1H-NMR spectroscopy (Fig. S-1) for monomer conversion and by SEC for molecular weight and dispersity. The respective results are summarized in table 1. The polymers were named P-38, P-92, P-435, with the numbers representing the respective degree of polymerization as determined from 1H-NMR spectra. For P-435, the targeted degree of polymerization is quite high for a RAFT polymerization38,

39

, consequently leading to poor

control and thus to a high polydispersity (PDI) as well as a strong deviation of the detected MW from the theoretical value. Nevertheless, the three synthesized polymers exhibit significantly different chain lengths, which is sufficient to study the influence of the chain length on the properties of the conjugates. Table 1. Specifications of all PNIPAm samples to be conjugated with DERA. The polymers were synthesized via RAFT using PDS-PABTC as CTA and ABCVA as initiator. The polymerizations were conducted in dioxane at 90 °C. Polymer [M]/[CTA] DPa) MnSEC b) PDI b) P-38

50

38 (4.7 kDa)

5.9 kDa

1.37

P-92

100

92 (10.8 kDa)

12.9 kDa

1.33

P-435

500

435 (49.6 kDa)

29.1 kDa

1.93

a)

calculated from the conversion (determined by NMR spectroscopy, see Fig S-1) assuming the absence of termination and transfer reactions. b) DMF-SEC with polystyrene standards.

Conjugation The two amino acids commonly used for the chemical modification of proteins are lysine and cysteine.31 One DERA molecule bears 4 cysteine and 19 lysine residues and both the cysteine and the lysine can in principle be used for the conjugate formation. However, as it is also a lysine residue (Lys167) that plays a major role in the catalytic mechanism40, utilizing lysines for the conjugation could lead to a strong decrease in enzymatic activity. From previous investigations,



we know that conjugation of lysine-reactive PNIPAm (Scheme S-2) to DERA indeed lowered the activity of the enzyme significantly (Fig. S-2). ‘Grafting from’ as well as ’grafting to’, which we use for the attachment of polymers, normally allows for an adjustment of the conjugation efficiency by variation of the stoichiometry of the reaction partners, thus enabling us to limit the decline of activity. However, a principle avoidance of a conjugation to Lys167 is still not possible. Hence, we decided to address the cysteine residues for conjugation, even though their abundance is relatively low (4 cysteine residues per DERA molecule) and thus the maximum number of chains that can be attached is limited. Consequently, the cysteine reactive group pyridyl disulfide was used for the conjugation of the polymer chains. The polymer with the protein reactive end group is mixed with DERA in a phosphate buffered solution (pH = 7.0) and is then, after purification, subjected to analysis. The conjugation process is depicted in Scheme 2. The PDS-PNIPAm chains with the different molecular weights were conjugated to DERA in a 1:1, 1:2, 1:5 and 1:10 cysteine/polymer end group molar ratio, respectively. After overnight reaction at 4 °C, the conjugates were analyzed by a thiol quantification assay and SDS-PAGE. The thiol quantification (Fig. S-3) reveals, that the conjugation efficiency reached a value of only 25 % with a 1:10 cysteine/polymer end group ratio, meaning that only 1 out 4 cysteines is equipped with a polymer chain. The results show also, together with SDS-PAGE (Fig. S-4), that the conjugation efficiency decreases even further with decreasing polymer/protein ratio, although the differences are small. Thus, for all further studies a 1:10 cysteine/polymer end group ratio was chosen in order to maximize the degree of conjugation.

Scheme 2. Conjugation of DERA and PDS-PNIPAm via the grafting-to approach.

During the process of conjugate preparation, we found that the synthesized PNIPAm dissolves slowly in the buffer solution used for conjugation, even at lower temperatures of around 4 °C. This is caused by a “salting-out” effect originating from the potassium phosphate in the solution


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which strongly lowers the LCST of the polymer.41 This could be the reason for the low conjugation efficiency. To tackle this issue, we used sodium thiocyanate (NaSCN) which, in contrast to potassium phosphate, is known from the Hofmeister series to exert a strong “saltingin” effect and therefore is capable of increasing the phase transition temperature of polymers.35, 42, 43

In the conjugation step, NaSCN was added to the protein solution together with PNIPAm.35 As

before, the PDS-PNIPAm of different molecular weights was conjugated to the DERA in a 1:10 cysteine/polymer end group ratio respectively. The reaction specifications for all the conjugates prepared are summarized in Table 2. We observed that the polymer dissolves quickly at 4 °C in the buffer in the presence of 1 M NaSCN. At the same time the cloud point of a diluted polymer solution (conc. 5·10-3 mg/mL) shifts from 32 °C to 39 °C as observed via temperature-dependent light scattering measurements (Fig. S-5). The conjugation efficiency of PNIPAm to DERA was now found to be significantly improved, reaching values of up to 83% according to the thiol quantification assay (Table 2). Generally, we observe that the polymer chain length has no influence on the conjugation efficiency. The SDS-PAGE results (Fig. 1) indicate conjugates of different molecular weights as originating from the use of PNIPAm with varying chain length. Although the silver staining is not precise enough to quantify the conjugation efficiency, it is still obvious that the conjugate which was formed in the presence of NaSCN is present in a higher fraction in the reaction mixture compared to the experiments without NaSCN. This can be concluded from the weaker DERA bands compared to the conjugate bands at higher molecular weight (MW) within the respective columns. It should be mentioned that the presence of the NaSCN has the potential to slowly denature the enzyme.44 However, the conjugation reaction could be finished before a significant denaturing effect was detectable, as will be discussed later. Subsequent purification by centrifugal ultrafiltration finally removed the NaSCN from the reaction solution. The enzymatic activity of the obtained conjugates was maintained after storage for at least two months (Fig. S-10).



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Table 2. Summary of conjugation trials of DERA with PDS-PNIPAma) including conjugation efficiencies and specific enzymatic activities. Enzyme/ Conjugated Polymer 1 M NaSCN c) Conjugation Specific b) Conjugate (see Table 1) efficiency enzymatic [%]d) activity [U/mL] DERA

-

-

-

0.58

DERA

-

+

-

0.51

C-38

P-38

-

27

0.55

C-92

P-92

-

25

0.58

C-435

P-435

-

25

0.57

C-38n

P-38

+

81

0.31

C-92n

P-92

+

83

0.31

C-435n

P-435

+

82

0.30

a) The cysteine/polymer ratio is 1:10. The concentration of the protein is 0.5 mg/mL (phosphate buffer). b) The term “conjugated polymer” refers to the polymer, which was used for the conjugation reaction (see table 1), respectively. c) ‘+’ presence of NaSCN; ‘-’ absence of NaSCN. d) The term “conjugation efficiency” refers to the percentage of converted cysteines (4 cysteines per DERA molecule). The results are extracted from the thiol quantification assay (see Exp. part).

Figure 1. SDS-PAGE of DERA and the respective conjugates of DERA with PNIPAm of different molecular weight (see table 1) prepared without additional salt (column 2-4) and in the presence of 1M NaSCN (column 5-7). M represents the Marker.

After the conjugation, the excess PNIPAm had to be removed. Since the DERA in use is equipped with a His-tag, as it is commonly done for recombinant proteins expressed in E.coli in order to be able to purify them by affinity purification45, a His-tag spin column was used for the


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polymer removal. With this His-select column we could achieve a recycle rate of 80% of the protein and 60% of the conjugate as determined by a BCA assay. After the conjugation and purification, temperature-dependent light scattering (DLS) was performed to study the cloud point of the conjugate. The derived count rate of the three samples as a function of temperature is shown in Fig. 2A. One can clearly detect a positive correlation between the cloud point and the mass fraction of DERA within the conjugate, the latter being only determined by the polymer chain length as conjugation efficiencies are not affected by the PNIPAm chain length. The conjugate can be regarded as a kind of block copolymer structure with the enzyme moiety constituting a hydrophilic entity that is capable of shifting the cloud point of the thermoresponsive PNIPAm block to a higher temperature, a phenomenon that is commonly known for responsive block copolymer structures.46 At the cloud point, the PNIPAm chains bound to the DERA become insoluble in water, forming collapsed, hydrophobic patches on the enzyme surface. This in turn leads to an aggregation of the conjugates that finally leads to a strongly increased scattering intensity above TCP. For all the samples under investigation, CONTIN plots were recorded at 40 °C (Fig. 2B). While C-38n has still not reached its cloud point, leading to the conjugates being almost exclusively in the unimer state, C-92n and C435n form much larger particles ranging from 350 nm (C-92n) to 1 μm (C-435n), depicting that the conjugates with longer polymer chains aggregate into larger sized particles. The reason for that is that the hydrophobic PNIPAm, which tends to aggregate into large particles are partially shielded by the hydrophilic DERA, which is of course less easy when the polymer fraction in the conjugate is higher, as it is the case for C-435n. In other words, the protein entity shows a stabilizing effect on the colloidal PNIPAm-aggregates.



Figure 2. DLS analysis of the synthesized conjugates (Table 2) in phosphate buffer. A) Derived count rate as a function of temperature. B) Size distribution (CONTIN) by volume at 40 °C. The concentration of each sample was set to 5·10-3 mg/mL.


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Enzymatic activity of the conjugates As the conjugates were synthesized with and without NaSCN, the activity measurement was also done for both series of conjugates. For the underlying reaction mechanism of the activity assay, please refer to Scheme S-3. In the absence of NaSCN, the activity of the enzyme is almost unaltered upon the conjugation. The conjugation efficiency is quite low as detected by the thiol quantification assay, meaning that each enzyme molecule carries only 1 polymer chain on average. This means, that the conjugated enzymes are probably not too much disturbed with respect to their natural conformation. A different picture evolves when the conjugation is done in the presence of NaSCN. Here, activity loss of up to 49 % is detectable. The presence of the salt alone without polymer being attached to the enzyme decreased the activity only by 15%. The rest of the activity loss is therefore due to the attachment of the polymer chains. As the presence of NaSCN enhances the conjugation efficiency, consequently, the activity loss is higher compared to the conjugation trials where NaSCN was absent. Still, a retained activity of 50 % with respect to the initial value is a fairly good result (Fig. 3), considering the fact that the conjugation of polymer chains constitutes a significant disturbance in the natural conformation of the enzyme. As for the conjugation efficiency, the variation of the molecular weight of the polymers has almost no influence on the conjugate activity. Only for the highest chain length the activity has decreased a bit further. The effect, however, is not very pronounced. The most influential factor on the activity upon conjugation therefore seems to be the chemical conversion of cysteine residues and not the steric influence of the polymer chains. This makes sense if one takes into account that the available cysteine residues in DERAEC are not found near the catalytically active center (Fig. S-6). While this statement seems to contradict the fact that one cysteine (Cys47) is located close to the active center and can even intervene in the catalytic process17 (Scheme 3), it should be stressed, that the conjugation efficiency never exceeded 82 %. Thus, 1 out of the 4 cysteines per enzyme molecule is not converted, if we account for the experimental error in the cysteine quantification assay. Therefore, this one cysteine, most likely Cys47, may be considered as generally not available for conjugation.



Figure 3. Retained enzymatic activity after the conjugation step. The conjugation was either done with, or without the presence of NaSCN (Table 2). Cysteine/polymer end group ratio was always 1:10. The column inscriptions refer to the polymer which has been used, respectively (Table 1).

Acetaldehyde tolerance and recyclability of the conjugates As mentioned earlier, DERA is prone to deactivation in the presence of higher amounts of the industrial relevant substrates acetaldehyde and chloroacetaldehyde.47 One reason is a crotonaldehyde-derived Schiff base on Lys167, which can be formed by the aldol condensation product of acetaldehyde. By Michael type addition of Cys47 to this species, the active center is irreversibly blocked (Scheme 3).17 Consequently, we subjected our conjugates to a mixture of the two aldehyde compounds in order to assess conjugate stability in comparison with the unmodified enzyme. The activity measurement shows that unmodified DERA loses around 96 % activity when being exposed to the substrate mixture for 30 min. After 3 h, even more than 99 % activity is lost. On the other hand, the activity loss for the conjugates is not higher than 15 %, even after 3 h of exposure to the substrate mixture (Fig. 4). Again, different polymer chain lengths make no obvious difference. The conjugates are far more active than the unmodified DERA in the presence of a considerable amount of the industrially relevant aldehyde mixture. As a consequence to this, the conjugates can also be recycled from the activity assay multiple times (Fig. S-9), despite the fact that the retro aldol reaction utilized for the assay produces acetaldehyde as product. Conjugation as a means to improve the stability of an enzyme is widely used1,

3, 48

, e.g. to increase substrate tolerance, storage stability, heating stability, etc. The

conjugation of PNIPAm to DERA constitutes now another example of such a system. Note that


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the conjugates have a lower initial activity compared to the unmodified enzyme. A correlation between decreased initial activity and an increased stability towards acetaldehyde derivatives is a commonly observed feature for DERA, as a lower activity is expressed by a lower turnover rate and thus, a lower probability of crotonaldehyde formation and accumulation.17 This phenomenon, however, is not always observable, which we could prove by additional experiments. We also measured the tolerance of the conjugates with the lower conjugation efficiency as well as the tolerance of an enzyme that was not functionalized with polymer but with a low molecular weight compound that was initially designed to serve as ATRP type initiator for grafting-from conjugation (Scheme S-4 and S-5, Supporting Information). The polymer/enzyme conjugates exhibit a functionalization degree of only 25 % leading to an initial activity close to the value of the unmodified DERA. The loss of more than 95 % of activity after exposure to the aldehyde mixture is in line with our observations for the non-conjugated enzyme (Fig. S-8). However, the enzyme equipped with the low molecular weight ATRP initiating moiety, which shows a functionalization degree of around 50 % and is considerably less active than the unmodified DERA, still loses most of its activity upon the aldehyde tolerance test. Therefore, the lower initial activity may be the reason for the absence of crotonaldehyde accumulation and thus for the increased aldehyde tolerance of the enzyme, yet it is not a guarantee for it. In this light, the increased aldehyde tolerance of our conjugates does not appear to be self-evident.

Scheme 3. Reaction mechanism leading to acetaldehyde induced inactivation of DERA. Crotonaldehyde, which is the result of a side reaction during the conversion of the substrate, is irreversibly converted in a Michael addition of C4717 while being bound as Schiff base to Lys167.



Figure 4. Effects of acetaldehyde and chloroacetaldehyde on the specific enzymatic activity of DERA and its respective conjugates. A) Absolute activities as a function of aldehyde exposure time B) Relative activities as a function of aldehyde exposure time. The molar concentrations of acetaldehyde and chloroacetaldehyde were set to 300 mM and 150 mM, respectively. Before measurement, the solutions were purified via centrifugal ultrafiltration, respectively. The open triangle represents C-435n in immobilized form (sample B, table 3) as described in the section “film formation”.

Interfacial activity of the conjugates Pendant drop tensiometry is a simple and versatile method to measure interfacial tension (IFT). Consequently, it has been frequently employed for accessing the interfacial activity of various biomacromolecules.49 Dynamic interfacial tension experiments were carried out for DERA, PNIPAm and the DERA-PNIPAm conjugates in order to understand the dynamics of stabilization of the air/water interface by all the three kinds of species. As the conjugates were designed to form a thin film at the air-water interface later on, the measurement of the interfacial


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tension was done by the pendant drop method using a respective air/water system. Not surprisingly, the buffer by itself showed almost no interfacial activity (Fig. 5), as it contains the buffering phosphate salt. DERA, however, turned out to be significantly surface active (Fig. 5A). DERA has only a small hydrophobic patch on its surface which is not sufficient to induce a strong amphiphilicity.24 Still, DERA, like many water-soluble proteins, adsorbs to the air-water interface of the liquid drop within a certain time frame24, which is mostly the result of an unfolding of the protein.50-53 The pure PNIPAm samples show faster adsorption kinetics than DERA (Fig. 5B-D) with the IFT curves appearing similar for all three molecular weights (MW). Only for the samples with the lowest concentrations, an influence of the MW is detectable with P-38 showing the slowest IFT drop. The interfacial tension measurements of the conjugates C-38n, C-92n and C-435n revealed a remarkable adsorption behavior (Fig. 6). For C-38n and C-92n, the IFT curves are almost identical for the low and the medium concentration (2·10-4 and 2·10-3 mg/mL) respectively, with the values at 1000 s being a bit lower for C-92n than for C-38n. C-435n on the other hand shows increased adsorption kinetics already for the medium concentration. A further concentration increase to 2·10-2 mg/mL shows a strongly accelerated IFT drop for all three conjugates with C-92n and C-435n being almost instantly at a steady state. This concentration dependent strong increase in adsorption kinetics is in contrast to the pure DERA and the pure polymer, where these changes occur more gradually and steadily. This leads to a situation in which the conjugates show similar or even slower dynamics compared to the pure polymer at lower concentration but an opposite behavior at high concentration. Note, that this effect is least pronounced for the conjugates with the lowest polymer fraction (C-38n). Contributions to the surface activity of the conjugates therefore seems to come more from the polymer side than from the DERA which is in line with behavior observed for the pure compounds. The strongly increased dynamics at high concentration, however, only occurs for the conjugates and can therefore be considered a synergistic effect originating from the conjugation. Similar observations have been made before.29, 30 By investigating conjugates of PNIPAm and ferritin, it was concluded that PNIPAm attached to a protein is in a confined state and cannot move freely anymore, thereby being unable to adopt the usual globular structure at the interface where only a small segment of the polymer chain is adsorbed at the interface.30 In the conjugate the



translational freedom of the chains is severely restricted causing the chains to adopt a more stretched conformation and hence to occupy much more space at the interface. Consequently, the conjugates show an increased surface activity compared to the pure PNIPAm.

Figure 5. Interfacial tension as a function of time for the pure conjugate components measured by pendant drop tensiometry with air as ambient phase A) DERA. B) P-38. C) P-92. D) P-435.


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Figure 6. Interfacial tension as a function of time for the conjugates measured by pendant drop tensiometry with air as ambient phase. A) C-38n. B) C-92n. C) C-435n.

Film formation and activity of the film As the interfacial tension measurement shows that the three conjugates C-38n, C-92n and C435n exhibit significant interfacial activity, which exceed the value for pure DERA, film formation at an air-water interface should work straightforward. The conjugates are able to selfassemble at the air-water interface, thus forming a film that is settling on a suitable support after water evaporation. One drop of the conjugate solution was deposited on a silicon wafer, which was cleaned by a high pressure CO2 stream and activated by oxygen plasma before. Then, after the evaporation step, the thickness of the film was estimated by atomic force microscopy (AFM). To study the influence of the solution concentration of the conjugate on the film formation,



protein concentrations of 1·10-1 mg/mL, 2·10-2 mg/mL and 4·10-3 mg/mL were chosen, keeping the volume of the deposited droplet always at 120 µL (table 3). Table 3. Sample specifications for the self-assembly and film formation step including assessment of the specific enzymatic activities Conjugate Sample Conc.a) during Specific enzymatic activity [U/mL] (see Table 2) film formation In solution Immobilizedb) After [mg/mL] resolubilization DERA A 0.1 0.58 0.11 0.081 (non-conjugated, B 0.02 0.081 0.081 pure enzyme) c) C 0.004 - c) C-38n A 0.1 0.31 0.081 0.097 B 0.02 0.081 0.081 C 0.004 - c) - c) C-92n A 0.1 0.31 0.15 0.16 B 0.02 0.15 0.15 C 0.004 - c) - c) C-435n A 0.1 0.3 0.16 0.16 B 0.02 0.16 0.16 C 0.004 - c) - c) a) Concentration of enzyme/conjugate within the droplet (120 µL) that is deposited on the respective wafer. b) Instead of adding DERA or conjugate to the assay solution, a DERA/conjugate covered 1 x 1 cm glass slide was immersed, which carries the same amount of enzymatically active species as was present for the measurement in solution. c) No reliable data available.

For C-38n, the conjugates seem to aggregate on the silicon wafer but do not generate a homogeneous, closed film at any of the three concentrations (Fig. 7A). C-92n, immobilized with a concentration of 0.02 mg/mL in the deposited droplet, forms a film with a thickness of approx. 50 nm which indicates that a nano-thin macroscopic film could be indeed generated from the conjugate C-92n at an air/water interface. For the same sample deposited at a concentration of 0.004 mg/mL the difference of height between the scratched and the non-scratched area (see Exp. part) is not obvious, meaning that there is no continuous film of detectable thickness. Thus, again no real film has formed for this particular sample. For C-435n, all concentrations led to smooth films, the thickness of the film being much lower than the one detected for C-92n at the same concentration. For instance, the sample generated at 1·10-1 mg/mL forms a film with a thickness of approx. 10 nm while that of 2·10-2 mg/mL leads to a 4 nm thick film. This is particularly remarkable if one considers the fact that the depicted concentrations refer to the amount of enzyme and not the overall amount of conjugate material, meaning that for identical


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concentrations, solutions of C-435n contain even more solid material than solutions of C-92n. Yet, the films appear to be thicker for the latter. In general, one can conclude that a higher fraction of polymer leads to better film formation, while the film thickness decreases at the same time. The flexibility of the polymer chains facilitates the entanglement of single conjugate molecules, thus explaining the ability to form continuous films. The flattening of the film that occurs with increasing polymer fraction on the other hand is a result of drying effects in combination with an altered wettability. For C-92n, the detected thicknesses do more or less fit to the amount of spread material if one assumes a uniform distribution of material over the whole wafer surface. For C-435n on the other hand, the films appear way too thin for such an assumption. Indeed, visual inspection of the respective wafers indicates a non-uniform material distribution with more material being present at the rim. Nevertheless, large areas within the middle of the wafer are covered with a defined film of constant thickness.



Figure 7. AFM analysis of the films formed via self-assembly of the conjugates at the interface of respective buffer droplets that have been deposited on silicon wafers and left for drying overnight. A) Topography images. B) crosssectional analysis. For the sample specifications, please refer to Table 3.


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As a final step, the enzymatic activity of the generated films was investigated (Table 3). As the activity assay is done by measuring the absorbance of NADH, a transparent support is needed for the sample preparation. Therefore, the silicon substrate is replaced by glass. Besides that, the preparation of the film otherwise was identical to the AFM samples. For the assay, the film covered glass slide was simply immersed in the reagent solution within the well of a microtiter plate (the same as has been used for the assay of the conjugate in solution), followed by recording of the absorbance. The immersion was done carefully in order to ensure that the film is not accidently removed from the slide. The chosen sample concentrations for the film formation are the same as for the preparation of the AFM samples and again are referring to the protein concentration. The results are shown in Figure 8. First of all, the results for the lowest concentration of 4·10-3 mg/mL suffered from poor reproducibility owing to the low rate of NADH conversion which is already close to the rate observed for the auto decomposition of NADH. Therefore, we will only discuss the two higher concentrations. The film activity measurement results depict that the films made from the conjugates C-92n and C-435n show higher activities than the pure DERA film, although the conjugates have been less active in solution compared to the pure enzyme. In fact, at a film preparation concentration of 1·10-1 mg/mL and 2·10-2 mg/mL the C-92n and C-435n films retain app. 60 % and the C-38n film app. 30 % of their respective solution activity, while the DERA film retains only 15 %. The polymer chains therefore must have a stabilizing effect during adsorption of the conjugates at the interface and/or upon the deposition of the film on the glass substrate, thereby limiting the extent of conformational changes of the enzymatic structure during the whole immobilization process.3 As mentioned before, DERA is likely to unfold when adsorbing to an interface, thereby losing its activity to some extent. Such an unfolding event is also the main reason behind the surface activity of most globular, water soluble proteins.54 For the conjugate, this unfolding seems to be suppressed to a certain extent, most likely due to the fact that surface activity is already established by the attachment of the PNIPAm chains, rendering the driving force to unfold much weaker. The effect apparently is less pronounced for C-38n, where the polymer fraction is also rather low. Next to the assessment of film activity, respective measurements were also done after the film material has been washed off from the glass slide again. The results are also shown in Figure 8. Control experiments with the blank glass slides remaining from the washing step revealed residual apparent activities on the slide of 0.03 U/mL or less (which again is close to the NADH auto decomposition rate), verifying that most of the material has been washed off. The activity of the washed off material, now again in solution, is quite similar to the activity of the original film for all samples, meaning that somewhat decreased activity upon film formation and deposition can be attributed to an irreversible conformational change upon



the film building and deposition process. It is therefore not a result of a reversible disturbance of the enzymatic structure induced by enzyme/glass support interactions. It is noted that some of the enzyme containing material is redissolved already upon the activity assessment of the film (i.e. before the washing step), as indicated by a reduced activity of the film on the slide after it has been immersed into the activity assay solution and taken out again. This residual activity, however, which is in the range of 30-40 % indicates that a significant amount of film material remains on the slide throughout the duration of the activity measurement. Furthermore, we noticed that it requires several washing cycles to completely remove all material from the slide. Taking further into account that the specific activity before and after the washing step are more or less the same for all samples, although obviously both immobilized and solubilized material contribute to the activity before the washing step, it can be concluded that immobilized and solubilized enzyme have the same specific activity. This finding also leads us to the conclusion, that there is no diffusion limitation which would slow down the catalytic process, thus again emphasizing the benefit of a low film thickness. As a final remark it is noted that the overall picture discussed in this section is not changing when the activity assays were performed one week after sample preparation and not right away, meaning that the immobilized conjugates are fully stable over this time period, even if they are stored in dry state. Furthermore, the tolerance towards acetaldehyde is not affected upon immobilization (Fig. 4).

Figure 8. Relative, specific enzymatic activities of DERA and the conjugates at different stages of an immobilization/resolubilization process. The column inscriptions refer to the respective sample that has been investigated and ‘A’ and ‘B’ to the different immobilization conditions (see Table 3).

CONCLUSION


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In conclusion, we were able to equip 2-Deoxy-D-ribose-5-phosphate aldolase with PNIPAm chains of different chain lengths in a grafting-to approach by addressing the cysteine residues of the protein. We showed that the conjugation efficiency reached a value of around 80 % independent of polymer chain length, which lead to conjugates that bear 3 chains per enzyme molecule on average. Enzymatic activity dropped to half of the initial value, however, it came along with a strongly increased stability towards a mixture of acetaldehyde/chloroacetaldehyde. Conjugation also led to an increased surface activity of the enzyme with a synergistic effect being most pronounced for higher concentrations and polymer chain lengths. The polymer chain length also had an influence on the film building ability of the conjugates and the thickness of the resulting film during the self-assembly step at the air/water interface. Longer polymer chains and therefore a higher fraction of polymer in the conjugate lead to better defined but also thinner films. These effects are attributed to an enhanced spreading of the PNIPAm chains and an improved entanglement of the conjugates at the interface. Finally, activity studies of the films proved that the conjugation has a stabilizing effect during the self-assembly of the conjugates, significantly limiting the activity loss upon adsorption of the conjugate at the interface. In addition, no difference in specific activity was detected between the immobilized and the dissolved conjugate pointing to the absence of any reaction retardation due to diffusion limitation. The successful immobilization of DERA in an ultrathin film constitutes a first step in the development

of

a

biocatalytic

synthetic

process

for

enantiomerically

pure

β-

monohydroxyaldehydes and β,δ-dihydroxyaldehydes in which product accumulation and in turn irreversible enzyme deactivation can be avoided by coupling the biocatalytic conversion with transport processes. In a follow-up work, the DERA containing films will be crosslinked and deposited on porous supports that in turn will be implemented into membrane test stations. With this set-up, DERA catalyzed, continuously operated synthesis processes can be performed with which enzyme kinetics but also operational stability and recyclability of the enzyme will be investigated.



Page 24 of 45

EXPERIMENTAL Materials The pyridyl disulfide equipped 2-propanoic acid butyl trithiocarbonate chain transfer agent (PDS-PABTC or PDS-CTA) was synthesized according to a procedure described elsewhere.35 The

synthesis

of

N-(α-bromoisobutyryloxyethyloxyethyl)maleimide

is

also

described

elsewhere.55 N-Isopropylacrylamide (NIPAm) (Sigma-Aldrich, 99%) was recrystallized from a mixture of n-hexane/toluene (4:1 v/v). Dipotassium hydrogenphosphate trihydrate (SigmaAldrich, ≥99.0%), potassium dihydrogenphosphate (Sigma-Aldrich, ≥99%), 4,4’-azobis(4cyanovaleric acid) (ABCVA) (Sigma-Aldrich, ≥75%), CDCl3 (Roth, 99.8% D), bovine serum albumin (BSA) (Sigma-Aldrich, ≥98%, lyophilized powder, heat shock fraction), sodium thiocyanate (Sigma-Aldrich, ≥98%), sodium dodecyl sulfate (Roth, ≥99.5%), methyl maleimidobenzochromenecarboxylate (MMBC/ ThioGlo-1, Berry & Associates Inc.), RotiLoad® 3 (LDS) Protein loading buffer, non-reducing (Roth, 4×conc.), 2-deoxyribose 5-phosphate sodium salt (DRP) (Sigma-Aldrich, ≥95%), NADH disodium salt (Roche, ~100%, Grade I), α-glycerophosphate dehydrogenase-triosephosphate Isomerase from rabbit muscle (SigmaAldrich, Type III, ammonium sulfate suspension, TPI 750-2000 units/mg protein, GDH 75200 units/mg protein (biuret)), acetaldehyde (Sigma-Aldrich, ≥99.5%) and chloroacetaldehyde (Sigma-Aldrich, ~50 wt% in H2O) were used as received. Silicon wafers (p-type, 625 µm thickness, front side polished) were purchased from CrysTec, glass slides (cut edges) from VWR. The Pierce™ BCA protein assay kit was purchased from ThermoFisher scientific. Buffers The pH of all buffers used was controlled and adjusted using a Mettler Toledo Seven CompactTM pH-meter that is calibrated with pH 4, 7 and 9 calibration standards (Mettler Toledo). If not stated otherwise, the term phosphate buffer refers to 20 mM of potassium phosphate in Milli-Q with a pH adjusted to 7.0. Production of 2-deoxy-D-ribose-5-phosphate aldolase from Escherichia coli (DERAEC) DERAEC was expressed in E.coli and purified by Nickel charged affinity resin (NiNTA) and desalting columns (PD10) according to Dick et al.56 Before lyophilization the protein was dissolved in KPi-buffer at 20 mM and pH 7 and set to a concentration of ca. 5 mg/mL. Synthesis of end-group functionalized poly(N-isopropylacrylamide) (PNIPAm) for protein conjugation


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For the RAFT polymerization of NIPAm, a pyridyl disulfide equipped RAFT agent based on PABTC was used. The NIPAm: CTA: ABCVA molar ratio was set to 100:1:0.1, 20:1:0.1 and 500:1:0.1, respectively. In a typical procedure, NIPAm (10 mmol, 1.13 g), CTA (0.1 mmol, 40.7 mg) and ABCVA (0.01 mmol, 2.80 mg) were transferred into a schlenktube and dissolved in dioxane (5.65 mL). After three freeze-pump-thaw cycles, the schlenktube was refilled with nitrogen. Then the solution was heated to 90 °C in an oil bath for 3 h before being quenched by cooling in an ice water bath and subsequent exposure to air. The polymeric product was precipitated into diethyl ether and dried under reduced pressure to yield PNIPAm with a protein reactive chain end. 1

H-NMR

1

H-NMR was recorded on a Bruker 500 MHz FT-NMR spectrometer using CDCl3 as solvent.

Size exclusion chromatography (SEC) SEC analysis was done on a device using DMF (with 0.1 % LiBr) as eluent using PS standards. The sample was passed over a combination of the columns PSS GRAM Guard (8 x 50 mm), PSS GRAM 1000 Å (300 x 7.5 mm), PSS GRAM 1000 Å (300 x 7.5 mm) and PSS GRAM 30 Å (300 x 7.5 mm) with a flow rate of 1.0 mL/min. Detection was achieved with a refractometer SEC3010 (WGE Dr. Bures) and analysis was performed with the Software Parsec 5.62 (Brookhaven Instruments). Conjugation of PNIPAm to DERA In a typical procedure, a solution of DERA (1.74 μM, 0.5 mg/mL) was prepared in phosphate buffer in which the respective PNIPAm has been dissolved before. The ratio of cysteine/protein reactive polymer end group (4 mol cysteines per mol DERA) was set to 1:1, 1:2, 1:5 and 1:10 respectively for each polymer. For the conjugation reactions in the presence of NaSCN, a 1 M solution of NaSCN in phosphate buffer was used instead of pure buffer to dissolve the reactants. Here NaSCN and PNIPAm were mixed and transferred together into a centrifugal tube to which the

buffer

was

added.

Next,

the

mixture

was

incubated

overnight

at

4 °C with continuous shaking in a thermal mixer (Compact thermal mixer, Eppendorf). Conjugation efficiency was evaluated by a thiol quantification assay and SDS-PAGE (see below). His-select spin columns (Sigma-Aldrich) were used to separate the conjugate from the excess free polymer. 600 µL of equilibration buffer (50 mM sodium phosphate with 0.3 M sodium chloride, pH 8.0) was added to the spin-column followed by centrifugation (centrifuge 5424R,



Eppendorf) at 4000 rpm at room temperature for 2 min. Then the samples were loaded onto the column. After another centrifugation at 4000 rpm at room temperature for 2 min, the flowthrough was collected, reloaded onto column and centrifuged another time. This collectionreload-centrifugation step was repeated for 5 times. Finally the elution buffer (50 mM sodium phosphate with 0.3 M sodium chloride and 250 mM imidazole, pH 8.0) was loaded onto the column followed by another centrifugation analogue to the step above. The collected solution fractions contained the purified conjugates. BCA assay The BCA assay for determination of protein concentration was performed with the help of a microplate reader (Infinite M200 Pro, Tecan). The sample was replicated into wells of a transparent 96-well plate (Thermo Scientific Pierce). Working reagent was then added to the well followed by incubation at 37 °C for 30 min. In case of the conjugates, incubation was done at room temperature for 2 h. After incubation, the absorbance was recorded at 562 nm. Thiol quantification assay The thiol quantification assay was performed with the help of a microplate reader (Infinite M200 Pro, Tecan) using black 96-well plates (Thermofisher scientific). 100 µL of sample (conc. 0.5 mg/mL) and 100 µL phosphate buffer with 2 wt% SDS were added to a well. Then 2 µL of ThioGlo-1 stock solution in acetonitrile (1.5 mM) was added. The mixture was then incubated for 2 h at room temperature followed by recording of the fluorescence intensity. (λEx = 379 nm, λEm = 513 nm). As a standard, a solution of BSA of known concentration was used. Gel electrophoresis (SDS-PAGE) DERA and conjugates were analyzed on polyacrylamide gels (12 % bisacrylamide) at 90 V, 18 mA and 90 min per gel. Samples were prepared before by denaturing the protein using Roti®Load 3 protein loading buffer at 92 °C for 10 min. Then, 8 mL of each sample at the concentration of 0.5 mg/mL was loaded onto the gel. Silver (with Roti®-Mark BICOLOR from Carl Roth, Germany as the marker) staining was used to develop the gel. Centrifugal ultrafiltration of enzyme/conjugates Purification and recycling of the samples by centrifugal ultrafiltration was done with 0.5 mL centrifugal filter units (Amicon®) equipped with a membrane made from regenerated cellulose (MWCO: 10 kDa). The sample was loaded to the units followed by centrifugation at 10000 rpm for 10 min (centrifuge 5424R, Eppendorf) each cycle.


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Dynamic light scattering (DLS) Temperature dependent DLS measurements were done on a Malvern MAL1083122 Zeta-sizer using disposal cuvettes (ZEN 0040). The instrument uses a 633 nm ‘red’ laser and the detector position is 173°. At each temperature 3 measurements were performed and averaged respectively with automatic adjustment of measurement duration. Temperature was changed in steps of 1 °C with an equilibration time of 120 s before measurement. The sample concentration was set to 5·10-3 mg/mL (phosphate buffer) in each case. Enzyme activity assay The activity assay, based on the retro aldol type cleavage of DRP was determined by following the oxidation of NADH using a coupled assay that involves the reduction of the retro aldol product by TPI and GPD20 (see Supporting Information). In the standard set-up, the reaction mixture was composed of phosphate buffer (0.1 M, pH 7), 1.5 mM DRP, 0.15 mM NADH, 1 U of TPI, 10 U of GPD and 0.5 mg/mL DERA or conjugates. The volume was set to 1 mL in each case. The change in absorbance of NADH was assayed in 24-well plates at 340 nm. In the case of the immobilized enzyme/conjugates, instead of adding DERA or conjugate to the assay solution in a solubilized form, a 1 x 1 cm glass plate carrying a film of the respective enzymatically active species (film preparation see below) was immersed into the assay solution within the respective well. To investigate the activity of the enzyme after resolubilization, the glass slide carrying the immobilized material was rinsed several times with phosphate buffer (0.1 M, pH 7) followed by collection of the single fractions and a reconcentration step by centrifugal ultrafiltration. The resulting enzyme/conjugate containing solution was used for the activity measurement as described above. The volume activity of DERA solution is calculated from the following formula (V = test volume = 1000 µL; ε = 6.22 L mmol-1cm-1; ν = sample volume = 10 µL; d = 1 cm; f = dilution factor of enzyme solution). ∆‫ܣ‬ ܸ ܷ ∆‫ܣ‬ ݉݅݊ = ݂= 16.1݂ ݉‫ܮ‬ ߝߥ݀ ݉݅݊

Acetaldehyde tolerance To examine the effects of the industrial relevant substrates acetaldehyde and chloroacetaldehyde on enzyme stability, enzyme and conjugate solutions in phosphate buffer were incubated for time intervals in the range of 30 to 180 min at 4 °C in the presence of 300 mM acetaldehyde and



150 mM chloroacetaldehyde, respectively. After removal of the substrates from the respective solution by centrifugal ultrafiltration (see above) the enzymatic activity of each sample was determined using the enzyme activity assay (see above). Interfacial activity Interfacial tension was measured on a Dataphysics OCA 15 EC device equipped with a CCD video camera having a resolution of 752 × 582 pixels. Interfacial tension was estimated by fitting the Young–Laplace equation to the image of the droplet in an inverted view. The droplet was generated from the respective protein, polymer or conjugate solution while the ambient phase was air. Dynamic tracking was used to collect data every two seconds for a droplet volume of 15 mL and the resulting value of interfacial tension was plotted against time. Film preparation by self-assembly at the air/water interface 1 x 1 cm silicon wafers (for AFM studies) or glass slides (for the activity assay) were first cleaned by ultrasonication in ethanol and then by snow-jet treatment. After blow-drying, they were treated with oxygen plasma (5 min, 0.2 mbar, 100 W, 100 % O2) using a PlasmaFlecto 10 Plasma Oven (Plasma Technology) equipped with an oxygen concentrator (525 Series, DeVilbiss). Then, 120 µL of enzyme or conjugate solution (enzyme conc. = 0.1 – 0.04 mg/mL) was drop casted onto the wafer/slide. Afterwards, the sample was left over night until all liquid had evaporated. Atomic force microscopy (AFM) AFM analysis was performed on a Bruker Dimension Icon using NanoScope 9.1 for the measurement and NanoScope Analysis 1.5 for image processing. Measurements were recorded in tapping mode using a Si tip with a spring constant of ~40 N/m and applying a scan rate of 1 Hz. A scratch was made on each film to expose the underlying silicon wafer. Then the thickness of film was estimated by scanning perpendicular to the scratch.


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ASSOCIATED CONTENT Supporting Information NMR analysis of the synthesized polymers, supplemental data on conjugation efficiencies and enzymatic activities, supplemental DLS data and background information on the enzyme structure and the activity assay as well as on synthetic procedures. The following files are free of charge. Supporting Information.pdf

AUTHOR INFORMATION Corresponding Author * E-Mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

FUNDING SOURCES This work was supported by the Ministry of Innovation, Science and Research within the framework of a seed fund research grant of the Bioeconomy Science Center Consortium of the federal state of North Rhine Westphalia, Germany (No. 313/323-400-002 13). Shuhao Zhang received a PhD scholarship from the China Scholarship Council.

ACKNOWLEDGEMENTS The authors thank Dr. Ulrich Glebe and Dr. Ruben Rosencrantz of the Fraunhofer Institute for Applied Polymer Research for the assistance in carrying out experiments and for giving helpful input during manuscript writing.

NOTES The authors declare no competing financial interest.



ABBREVIATIONS DERA - 2-deoxy-D-ribose-5-phosphate aldolase; NIPAm – N-isopropylacrylamide; BSA – Bovine Serum Albumin; LCST – lower critical solution temperature; PDS – pyridyl disulfide; CTA – chain transfer agent; IFT – interfacial tension

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Scheme 1. Formation of a catalytically active DERA containing thin film by self-assembly of respective DERA/PNIPAm conjugates. 182x146mm (96 x 96 DPI)



Scheme 2. Conjugation of DERA and PDS-PNIPAm via the grafting-to approach. 201x96mm (200 x 200 DPI)


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Figure 1. SDS-PAGE of DERA and the respective conjugates of DERA with PNIPAm of different molecular weight (see table 1) prepared without additional salt (column 2-4) and in the presence of 1M NaSCN (column 5-7). M represents the Marker. 137x83mm (96 x 96 DPI)



Figure 2. DLS analysis of the synthesized conjugates (Table 2) in phosphate buffer. A) Derived count rate as a function of temperature. B) Size distribution (CONTIN) by volume at 40 °C. The concentration of each sample was set to 5·10-4 mg/mL. 117x175mm (300 x 300 DPI)


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Figure 3. Retained enzymatic activity after the conjugation step. The conjugation was either done with, or without the presence of NaSCN (Table 2). Cysteine/polymer end group ratio was always 1:10. The column inscriptions refer to the polymer which has been used, respectively (Table 1). 114x79mm (200 x 200 DPI)



Scheme 3. Reaction mechanism leading to acetaldehyde induced inactivation of DERA. Crotonaldehyde, which is the result of a side reaction during the conversion of the substrate, is irreversibly converted in a Michael addition of C4717 while being bound as Schiff base to Lys167. 191x33mm (200 x 200 DPI)


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Figure 4. Effects of acetaldehyde and chloroacetaldehyde on the specific enzymatic activity of DERA and its respective conjugates. A) Absolute activities as a function of aldehyde exposure time B) Relative activities as a function of aldehyde exposure time. The molar concentrations of acetaldehyde and chloroacetaldehyde were set to 300 mM and 150 mM, respectively. Before measurement, the solutions were purified via centrifugal ultrafiltration, respectively. The open triangle represents C-435n in immobilized form (sample B, table 3) as described in the section “film formation”. 165x237mm (200 x 200 DPI)



Figure 5. Interfacial tension as a function of time for the pure conjugate components measured by pendant drop tensiometry with air as ambient phase A) DERA. B) P-38. C) P-92. D) P-435. 228x187mm (200 x 200 DPI)


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Figure 6. Interfacial tension as a function of time for the conjugates measured by pendant drop tensiometry with air as ambient phase. A) C-38n. B) C-92n. C) C-435n. 226x186mm (200 x 200 DPI)



Figure 7. AFM analysis of the films formed via self-assembly of the conjugates at the interface of respective buffer droplets that have been deposited on silicon wafers and left for drying overnight. A) Topography images. B) cross-sectional analysis. For the sample specifications, please refer to Table 3. 224x292mm (200 x 200 DPI)


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Figure 8. Relative, specific enzymatic activities of DERA and the conjugates at different stages of an immobilization/resolubilization process. The column inscriptions refer to the respective sample that has been investigated and ‘A’ and ‘B’ to the different immobilization conditions (see Table 3). 201x140mm (200 x 200 DPI)


Biocatalytically Active Thin Films via Self-Assembly ... - ACS Publications

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