Immobilization of Aldoxime Dehydratases and Their Use as Biocatalysts in Aqueous Reaction Media

: Immobilization of biocatalysts is a current topic in research enabling the easy recovery of catalysts from the reaction medium after the reaction, and it is often accompanied by a stabilization of the catalysts, which enables recycling. Within our ongoing research on the utilization of aldoxime dehydratases in the cyanide-free synthesis of nitriles through dehydration of readily available aldoximes, a screening of di ﬀ erent immobilization methods for free enzymes was performed. The applied immobilization methods are based on covalent binding and hydrophobic interactions of the enzyme with the carrier material and whole-cell immobilization in calcium alginate beads with and without subsequent coating. In our study, we found that the immobilization with puriﬁed free aldoxime dehydratases from OxdRE ( Rhodococcus erythropolis ) and OxdB ( Bacillus sp. strain OxB-1) leads to high immobilization e ﬃ ciencies, but also to a strong loss of activity with a residual activity of < 20%, regardless of the carrier material used. However, when using whole cells for immobilization instead of puriﬁed enzymes, we could increase the residual activity signiﬁcantly. Escherichia coli BL21(DE3)-CodonPlus-RIL OxdRE and OxdB whole cells were entrapped in calcium alginate beads and coated with silica using tetraethylorthosilicate (TEOS), leading to immobilized catalysts with up to 75% residual activity and a higher stability compared to the free whole cells. Even after three rounds of recycling, which corresponds to a 3 d reaction time, the immobilized OxdB whole cells showed a residual activity of 85%.


Introduction
Since biocatalysts have emerged as a valuable alternative to metal-and organo-catalysts [1][2][3][4][5][6], it is of major interest to develop highly efficient and stable biocatalysts, which in the best case can be reused. This can, for example, be realized by immobilization of the biocatalyst on a carrier or by entrapment in polymers or hydrogels. Such a heterogenized catalyst can be separated from the reaction medium more easily, usually by filtration. In comparison, whole-cell catalysts are often separated by centrifugation [7][8][9] or dissolved enzymes in an aqueous reaction medium which usually cannot be isolated. The free enzymes often lead to problems during the isolation of the product, especially if extraction is used. Extraction often leads to precipitation of the enzymes due to the denaturating effect of organic solvents, and the precipitate forms an interphase between the organic and aqueous phase [1][2][3][4][5][6][10][11][12]. This often makes downstream-processing tedious, thus causing lower isolated yields. In addition, in many cases, the biocatalyst cannot be reused after such an extractive work-up. Thus, for an improved process, a suitable immobilization method for the use of most biocatalysts is desirable. There are many different immobilization methods known, and different biocatalyst formulations can be used. On the one hand, free enzymes (used as crude extract or in the purified form) can be used, while Scheme 1. Immobilization strategies of enzymes.
The first strategy is heterogenization by crosslinking of the enzymes. The most prominent example are CLEAs (cross-linked enzyme aggregates) [13], which are formed by a preactivation of primary amino functionalities on the enzyme surface, such as the side chain of lysine, by glutaraldehyde and subsequent crosslinking of free primary amino groups with glutaraldehydebound amino groups to form heterogenized enzyme clusters. A second strategy of enzyme immobilization is a covalent binding of enzymes to carriers [14]. This can be performed, for example, by either usage of epoxy-carrier material, which reacts with side chains of amino acids of the enzyme surface, or by usage of amino carriers, which are preactivated by glutaraldehyde and react with amino side chains similar to the formation of CLEAs. A third technique is a strategy in which enzymes are bound non-covalently, for example, by hydrophobic interactions to carrier material with hydrophobic residues [14] or His-tagged enzymes on specific carriers [15], which often has the benefit of a weaker impact than covalent binding. Moreover, with free enzyme immobilization, whole-cell catalysts can also be immobilized. Many methods are known, for example immobilization of whole cells by entrapment in polymers, hydrogels, or other materials. To give an example for the entrapment of biocatalysts in polymers, von Langermann et al. developed a technique of whole-cell immobilization in polyurethane, which is a suitable heterogenized biocatalyst with a higher stability when compared to free whole cells [16,17,18]. Whole-cell catalysts (or free enzymes [19,20]) in an aqueous medium can also be immobilized by entrapment in superabsorber, yielding in a solid aqueous phase which can be used in an organic solvent as reaction medium and easily separated by filtration [21]. Although this technique allows biocatalytic reactions in organic solvents, it has the disadvantage that this immobilized biocatalyst cannot be used in aqueous medium, as it would lead to a leaching of the absorbed biocatalyst out of the superabsorber. Furthermore, the organic solvent usually has a deactivating effect on the enzymes; thus, the heterogenized catalyst cannot be reused [22]. Enzyme immobilization can also be performed by entrapment in calcium alginate beads. This immobilization technique was recently applied by the Patel group to combine a pig liver esterase with a Grubbs catalyst-both of which need different reaction mediums [23,24,25,26]. In this study, we focused on the immobilization of aldoxime dehydratase (Oxd), which has only been immobilized Scheme 1. Immobilization strategies of enzymes.
The first strategy is heterogenization by crosslinking of the enzymes. The most prominent example are CLEAs (cross-linked enzyme aggregates) [13], which are formed by a preactivation of primary amino functionalities on the enzyme surface, such as the side chain of lysine, by glutaraldehyde and subsequent crosslinking of free primary amino groups with glutaraldehyde-bound amino groups to form heterogenized enzyme clusters. A second strategy of enzyme immobilization is a covalent binding of enzymes to carriers [14]. This can be performed, for example, by either usage of epoxy-carrier material, which reacts with side chains of amino acids of the enzyme surface, or by usage of amino carriers, which are preactivated by glutaraldehyde and react with amino side chains similar to the formation of CLEAs. A third technique is a strategy in which enzymes are bound non-covalently, for example, by hydrophobic interactions to carrier material with hydrophobic residues [14] or His-tagged enzymes on specific carriers [15], which often has the benefit of a weaker impact than covalent binding. Moreover, with free enzyme immobilization, whole-cell catalysts can also be immobilized. Many methods are known, for example immobilization of whole cells by entrapment in polymers, hydrogels, or other materials. To give an example for the entrapment of biocatalysts in polymers, von Langermann et al. developed a technique of whole-cell immobilization in polyurethane, which is a suitable heterogenized biocatalyst with a higher stability when compared to free whole cells [16][17][18]. Whole-cell catalysts (or free enzymes [19,20]) in an aqueous medium can also be immobilized by entrapment in superabsorber, yielding in a solid aqueous phase which can be used in an organic solvent as reaction medium and easily separated by filtration [21]. Although this technique allows biocatalytic reactions in organic solvents, it has the disadvantage that this immobilized biocatalyst cannot be used in aqueous medium, as it would lead to a leaching of the absorbed biocatalyst out of the superabsorber. Furthermore, the organic solvent usually has a deactivating effect on the enzymes; thus, the heterogenized catalyst cannot be reused [22]. Enzyme immobilization can also be performed by entrapment in calcium alginate beads. This immobilization technique was recently applied by the Patel group to combine a pig liver esterase with a Grubbs catalyst-both of which need different reaction mediums [23][24][25][26]. In this study, we focused on the immobilization of aldoxime dehydratase (Oxd), which has only been immobilized in superabsorber to date. This limits the use of Oxd immobilizates in pure organic mediums. The purpose of this project was to broaden the usability of immobilized Oxds also to aqueous reaction systems by usage of other immobilization techniques.

Results
The investigation of suitable immobilization strategies for Oxds was performed with two different enzymes, namely OxdB from Bacillus sp. OxB-1 and OxdRE from Rhodococcus erythropolis, which already emerged as an efficient catalyst for aldoxime dehydration, especially for aliphatic substrates [21,27,28]. We focused on the dehydration of n-octanaloxime to n-octanenitrile for our standard activity assays, since this substrates is accepted by both enzymes. In the first step, we investigated the immobilization of isolated enzymes (purified by Ni-NTA (Ni-nitrilotriacetic acid beads) affinity chromatography) on the different carriers listed in Table 1. Depending on the carrier material, the immobilization is based on different interactions between the carrier and the enzyme. A schematic presentation of the interactions between immobilization carriers and enzymes is summarized in Scheme 2.
Catalysts 2020, 10, x FOR PEER REVIEW 3 of 19 superabsorber to date. This limits the use of Oxd immobilizates in pure organic mediums. The purpose of this project was to broaden the usability of immobilized Oxds also to aqueous reaction systems by usage of other immobilization techniques.

Results
The investigation of suitable immobilization strategies for Oxds was performed with two different enzymes, namely OxdB from Bacillus sp. OxB-1 and OxdRE from Rhodococcus erythropolis, which already emerged as an efficient catalyst for aldoxime dehydration, especially for aliphatic substrates [21,27,28]. We focused on the dehydration of n-octanaloxime to n-octanenitrile for our standard activity assays, since this substrates is accepted by both enzymes. In the first step, we investigated the immobilization of isolated enzymes (purified by Ni-NTA (Ni-nitrilotriacetic acid beads) affinity chromatography) on the different carriers listed in Table 1. Depending on the carrier material, the immobilization is based on different interactions between the carrier and the enzyme. A schematic presentation of the interactions between immobilization carriers and enzymes is summarized in Scheme 2. Carriers h1-h3 are hydrophobic resins that non-covalently immobilize proteins via physical adsorption. We chose h1, h2 and h3 as hydrophobic carriers because they differ in surface area (h1: >80 m 2 /g, h2: >750 m 2 /g and h3: > 90m 2 /g) and because of the pore sizes of the resins (see Table 1). Besides non-covalent immobilization, we also tested carriers which covalently bind proteins. We chose one amino carrier (a1), which has a short ethylene spacer between resin and amino functionality. The amino group is preactivated by glutaraldehyde, which afterwards can covalently Carriers h1-h3 are hydrophobic resins that non-covalently immobilize proteins via physical adsorption. We chose h1, h2 and h3 as hydrophobic carriers because they differ in surface area (h1: >80 m 2 /g, h2: >750 m 2 /g and h3: > 90m 2 /g) and because of the pore sizes of the resins (see Table 1). Besides non-covalent immobilization, we also tested carriers which covalently bind proteins. We chose one amino carrier (a1), which has a short ethylene spacer between resin and amino functionality. The amino group is preactivated by glutaraldehyde, which afterwards can covalently react with (for example) amino functions of the protein (Scheme 3a). Lastly, two epoxy carriers were tested. Here, we chose epoxy methacrylate (e1) and epoxy/butyl methacrylate (e2), which differ in the spacer length between resin and epoxy functionality and pore size. Epoxy resins react under formation of a covalent bond with (for example) amino functionalities of the protein (Scheme 3b). functionality. The amino group is preactivated by glutaraldehyde, which afterwards can covalently react with (for example) amino functions of the protein (Scheme 3a). Lastly, two epoxy carriers were tested. Here, we chose epoxy methacrylate (e1) and epoxy/butyl methacrylate (e2), which differ in the spacer length between resin and epoxy functionality and pore size. Epoxy resins react under formation of a covalent bond with (for example) amino functionalities of the protein (Scheme 3b).

Scheme 3.
(a) Protein immobilization on amino carriers after preactivation of the resin with glutaraldehyde and (b) protein immobilization on epoxy carriers. X-ray structure shown is the crystal structure of OxdRE [29].
After immobilization of purified OxdB and OxdRE on the different carriers, the immobilization efficiency and residual activity of the protein in comparison with the purified Oxds were determined. Immobilization efficiencies were measured by determination of protein concentration of the purified enzyme solution in phosphate buffer (PPB) used for the immobilization and of the supernatant after immobilization ( Figure 1).  After immobilization of purified OxdB and OxdRE on the different carriers, the immobilization efficiency and residual activity of the protein in comparison with the purified Oxds were determined. Immobilization efficiencies were measured by determination of protein concentration of the purified enzyme solution in phosphate buffer (PPB) used for the immobilization and of the supernatant after immobilization ( Figure 1). functionality. The amino group is preactivated by glutaraldehyde, which afterwards can covalently react with (for example) amino functions of the protein (Scheme 3a). Lastly, two epoxy carriers were tested. Here, we chose epoxy methacrylate (e1) and epoxy/butyl methacrylate (e2), which differ in the spacer length between resin and epoxy functionality and pore size. Epoxy resins react under formation of a covalent bond with (for example) amino functionalities of the protein (Scheme 3b).

Scheme 3.
(a) Protein immobilization on amino carriers after preactivation of the resin with glutaraldehyde and (b) protein immobilization on epoxy carriers. X-ray structure shown is the crystal structure of OxdRE [29].
After immobilization of purified OxdB and OxdRE on the different carriers, the immobilization efficiency and residual activity of the protein in comparison with the purified Oxds were determined. Immobilization efficiencies were measured by determination of protein concentration of the purified enzyme solution in phosphate buffer (PPB) used for the immobilization and of the supernatant after immobilization ( Figure 1).   was immobilized on the carriers. n-octanaloxime was used as a substrate to determine the activity of the immobilized enzymes in comparison with purified OxdB and OxdRE ( Figure 2).
Catalysts 2020, 10, x FOR PEER REVIEW 5 of 19 Immobilization efficiencies were found to be very high in all cases and for both proteins. These immobilization efficiency values between ~80-90% indicate that nearly all the protein in the solution was immobilized on the carriers. n-octanaloxime was used as a substrate to determine the activity of the immobilized enzymes in comparison with purified OxdB and OxdRE ( Figure 2). Residual activities after immobilization on different carrier materials were found to be in ranges of ~5-10% of the activity of purified OxdB or OxdRE. Especially in the case of OxdB, the carrier material showed a strong impact on the residual activity. In the cases of h1 and a1, the residual activity was still approximately 10% of the activity of purified enzyme, whereas the epoxy carriers e1 and e2 as well as h2 and h3 showed lower residual activities of approximately 5%. In the case of OxdRE, the impact of the carrier material was found to be weaker than OxdB. Only the epoxy resins Residual activities after immobilization on different carrier materials were found to be in ranges of 5-10% of the activity of purified OxdB or OxdRE. Especially in the case of OxdB, the carrier material showed a strong impact on the residual activity. In the cases of h1 and a1, the residual activity was still approximately 10% of the activity of purified enzyme, whereas the epoxy carriers e1 and e2 as well as h2 and h3 showed lower residual activities of approximately 5%. In the case of OxdRE, the impact of the carrier material was found to be weaker than OxdB. Only the epoxy resins e1 and e2 showed lower residual activities of~7%. The amino carrier a1 and the hydrophobic and macroporous carriers h1-h3 showed residual activities of~15-20% compared to the purified enzyme. Since h1 Catalysts 2020, 10, 1073 6 of 19 and a1 carriers showed acceptable results for OxdB and OxdRE, these two carriers were chosen for further experiments.
A storage stability assay was performed using purified OxdB and OxdRE enzymes and h1 and a1 immobilizates of both. Purified enzyme and immobilizates were stored at 4 • C for 6 and 32 d, and the activity was examined (Figure 3).
Catalysts 2020, 10, x FOR PEER REVIEW 6 of 19 e1 and e2 showed lower residual activities of ~7%. The amino carrier a1 and the hydrophobic and macroporous carriers h1-h3 showed residual activities of ~15-20% compared to the purified enzyme. Since h1 and a1 carriers showed acceptable results for OxdB and OxdRE, these two carriers were chosen for further experiments. A storage stability assay was performed using purified OxdB and OxdRE enzymes and h1 and a1 immobilizates of both. Purified enzyme and immobilizates were stored at 4 °C for 6 and 32 d, and the activity was examined (Figure 3). Relative activity RE h1 RE a1 RE pure The storage stability assay was performed in triplicates and the results show that the residual activity after different storage times at 4 °C for 6 d is often higher for the immobilized enzyme compared to that for the purified enzyme. In detail, the a1 immobilizates of OxdB are more stable, The storage stability assay was performed in triplicates and the results show that the residual activity after different storage times at 4 • C for 6 d is often higher for the immobilized enzyme compared to that for the purified enzyme. In detail, the a1 immobilizates of OxdB are more stable, while for OxdRE, h1 immobilization seems to be more beneficial. After 32 d of storage, however, the activity dramatically decreases in all cases to~20% or lower.
Following this, a cosolvent study was performed using purified OxdB and OxdRE enzymes and h1 and a1 immobilizates (Figure 4). The effects of immobilization on the stability of OxdRE and OxdB against organic solvents should be tested. Therefore, the purified enzymes or immobilizates were incubated for different times in different cosolvents (20% (v/v)) or PPB; afterwards, the activity of the conversion of n-octanaloxime to n-octanenitrile was determined.
Catalysts 2020, 10, x FOR PEER REVIEW 7 of 19 while for OxdRE, h1 immobilization seems to be more beneficial. After 32 d of storage, however, the activity dramatically decreases in all cases to ~20% or lower. Following this, a cosolvent study was performed using purified OxdB and OxdRE enzymes and h1 and a1 immobilizates (Figure 4). The effects of immobilization on the stability of OxdRE and OxdB against organic solvents should be tested. Therefore, the purified enzymes or immobilizates were incubated for different times in different cosolvents (20% (v/v)) or PPB; afterwards, the activity of the conversion of n-octanaloxime to n-octanenitrile was determined.  In this cosolvent study, the activity of beads or purified enzymes without incubation was normalized to a relative activity of 1, and all measured activities after incubation were compared to this value. We found that OxdB and OxdRE immobilized enzymes were not much more stable after incubation in different cosolvents than purified enzymes. Dimethylsulfoxide (DMSO) seems to be the most unsuitable solvent for both enzymes and acetonitrile (ACN) the most favorable, while methanol deactivates the enzymes more. Unfortunately, even after a short incubation time of 1 h, a decrease in activity is found in all cases. Even without any co-solvent, a strong decrease in activity, with ~65% residual activity for OxdB and ~50% for OxdRE, of the purified enzymes was observed after just 1 h. The immobilizates show a stabilizing effect for all solvents. However, the deactivation of DMSO is strong even when using immobilizates. Although the immobilization seems to have a stabilizing effect on the enzymes, the activity of the immobilizates is very low in comparison with the free enzymes (~20% residual activity). These low activities are even decreased by the usage of organic solvents, which makes the use of purified enzyme or the tested immobilizates unsuitable for application in organic synthesis.
Therefore, we further investigated immobilization of OxdB and OxdRE whole-cell catalysts. Our group already found that Oxd whole cells can successfully be immobilized in superabsorber and used in organic media very efficiently [22]. A disadvantage of this system is that superabsorberimmobilized cells cannot be used in aqueous reaction medium due to the leaching of cells out of the superabsorber. Another immobilization technique that is also suitable for the immobilization of whole cells is based on sodium alginate, which can be premixed in an aqueous solution with the cells of interest and dropped into CaCl2 solution which hardens alginate. When applying this immobilization technique to OxdB and OxdRE whole cells, first, we needed to perform a buffer screening because Oxds are normally used in PPB as buffer, which destabilize alginate beads. After we switched the standard PPB buffer to HEPES buffer, which showed similar activity to PPB, we immobilized OxdB and OxdRE whole cells in alginate beads. They were prepared by dropping a sodium alginate solution premixed with the whole-cell catalysts and buffer (HEPES, 50 mM, pH 7) into a CaCl2 solution to harden the beads. The beads were filtered, washed with HEPES buffer and used afterwards for biotransformation of n-octanaloxime to n-octanenitrile ( Figure 5). In this cosolvent study, the activity of beads or purified enzymes without incubation was normalized to a relative activity of 1, and all measured activities after incubation were compared to this value. We found that OxdB and OxdRE immobilized enzymes were not much more stable after incubation in different cosolvents than purified enzymes. Dimethylsulfoxide (DMSO) seems to be the most unsuitable solvent for both enzymes and acetonitrile (ACN) the most favorable, while methanol deactivates the enzymes more. Unfortunately, even after a short incubation time of 1 h, a decrease in activity is found in all cases. Even without any co-solvent, a strong decrease in activity, with~65% residual activity for OxdB and~50% for OxdRE, of the purified enzymes was observed after just 1 h. The immobilizates show a stabilizing effect for all solvents. However, the deactivation of DMSO is strong even when using immobilizates. Although the immobilization seems to have a stabilizing effect on the enzymes, the activity of the immobilizates is very low in comparison with the free enzymes (~20% residual activity). These low activities are even decreased by the usage of organic solvents, which makes the use of purified enzyme or the tested immobilizates unsuitable for application in organic synthesis. Therefore, we further investigated immobilization of OxdB and OxdRE whole-cell catalysts. Our group already found that Oxd whole cells can successfully be immobilized in superabsorber and used in organic media very efficiently [22]. A disadvantage of this system is that superabsorber-immobilized cells cannot be used in aqueous reaction medium due to the leaching of cells out of the superabsorber. Another immobilization technique that is also suitable for the immobilization of whole cells is based on sodium alginate, which can be premixed in an aqueous solution with the cells of interest and dropped into CaCl 2 solution which hardens alginate. When applying this immobilization technique to OxdB and OxdRE whole cells, first, we needed to perform a buffer screening because Oxds are normally used in PPB as buffer, which destabilize alginate beads. After we switched the standard PPB buffer to HEPES buffer, which showed similar activity to PPB, we immobilized OxdB and OxdRE whole cells in alginate beads. They were prepared by dropping a sodium alginate solution premixed with the whole-cell catalysts and buffer (HEPES, 50 mM, pH 7) into a CaCl 2 solution to harden the beads. The beads were filtered, washed with HEPES buffer and used afterwards for biotransformation of n-octanaloxime to n-octanenitrile ( Figure 5). Catalysts 2020, 10  The activity of OxdB whole cells and immobilized OxdB whole cells was determined. As shown in Figure 5, both whole-cell catalysts are still active after immobilization in alginate beads, however, the activity drops to approx. 60% in case of OxdB and approx. 20% in case of OxdRE in comparison with the free whole-cell catalysts.
Since the surface of calcium alginate is very hydrophilic and might negatively influence the diffusion of n-octanaloxime into the beads, we further investigated a coating of the beads with tetraethylorthosilicate (TEOS) (Scheme 4). The activity of OxdB whole cells and immobilized OxdB whole cells was determined. As shown in Figure 5, both whole-cell catalysts are still active after immobilization in alginate beads, however, the activity drops to approx. 60% in case of OxdB and approx. 20% in case of OxdRE in comparison with the free whole-cell catalysts.
Since the surface of calcium alginate is very hydrophilic and might negatively influence the diffusion of n-octanaloxime into the beads, we further investigated a coating of the beads with tetraethylorthosilicate (TEOS) (Scheme 4). Catalysts 2020, 10,  This coating is performed after immobilization of OxdB or OxdRE in alginate beads as previously described by stirring the beads overnight in a solution of TEOS in n-hexane. After this "silica-coating", the surface of the beads is more hydrophobic and the substrate may diffuse into the beads more easily. The silica-coated (Si-coated) beads were also tested for the conversion of noctanaloxime and compared to the results of free whole cells and uncoated alginate-immobilized whole cells ( Figure 5).
Indeed, the silica-coating of the beads seems to have an impact on the activity of the immobilized whole cells. For both enzymes, the residual activity is higher after silica-coating compared to the uncoated beads; however, the residual activity of OxdRE is still much lower when compared to OxdB (~20% residual activity for OxdRE and ~75% residual activity for OxdB). This finding is very surprising because Oxds (also in whole cells) are normally found to be unstable in organic media, while in this case, stirring of the beads overnight in pure n-hexane with TEOS leads to immobilized cells that remain active. Thus, this result motivated us to test the activity of the beads also in a pure organic solvent ( Figure 6). This coating is performed after immobilization of OxdB or OxdRE in alginate beads as previously described by stirring the beads overnight in a solution of TEOS in n-hexane. After this "silica-coating", the surface of the beads is more hydrophobic and the substrate may diffuse into the beads more easily. The silica-coated (Si-coated) beads were also tested for the conversion of n-octanaloxime and compared to the results of free whole cells and uncoated alginate-immobilized whole cells ( Figure 5).
Indeed, the silica-coating of the beads seems to have an impact on the activity of the immobilized whole cells. For both enzymes, the residual activity is higher after silica-coating compared to the uncoated beads; however, the residual activity of OxdRE is still much lower when compared to OxdB (~20% residual activity for OxdRE and~75% residual activity for OxdB). This finding is very surprising because Oxds (also in whole cells) are normally found to be unstable in organic media, while in this case, stirring of the beads overnight in pure n-hexane with TEOS leads to immobilized cells that remain active. Thus, this result motivated us to test the activity of the beads also in a pure organic solvent ( Figure 6). Figure 6. Dehydration of n-octanaloxime using immobilized whole cells in calcium alginate beads (alginate) or Si-coated calcium alginate beads (Si-beads) in pure organic medium compared to free whole cells in a biphasic reaction medium of OxdB (a) and OxdRE (b). In the y-axis an interruption from 25 to 95% is inserted.
It is already known that free Oxd whole cells lose their activity by usage in pure organic solvent or in a biphasic reaction medium [22]. For comparison reasons, we also applied free Oxd whole cells in a biphasic reaction medium compared to immobilized cells in alginate beads and Si-coated beads. As expected, we could not obtain any conversion in the biphasic approach in all tested solvents for free Oxd whole cells, namely cyclohexane (C), 2-methyl-tetrahydrofurane (T), methyl-tert-butyl ether (M) and n-hexane (H). In the case of the OxdRE immobilizates, we found conversion in cyclohexane as solvent; however, the conversions were very low and no difference between the coated and the uncoated beads were observed. In the case of OxdB immobilizates we could reach ~10% conversion by usage of cyclohexane as solvent for Si-coated beads. Even with the uncoated beads, a conversion of ~5% was observed. As we used n-hexane as solvent for TEOS coating, we further investigated nhexane as solvent and found a conversion of 20% by using Si-coated OxdB immobilizate. This result  Figure 6. Dehydration of n-octanaloxime using immobilized whole cells in calcium alginate beads (alginate) or Si-coated calcium alginate beads (Si-beads) in pure organic medium compared to free whole cells in a biphasic reaction medium of OxdB (a) and OxdRE (b). In the y-axis an interruption from 25 to 95% is inserted.
It is already known that free Oxd whole cells lose their activity by usage in pure organic solvent or in a biphasic reaction medium [22]. For comparison reasons, we also applied free Oxd whole cells in a biphasic reaction medium compared to immobilized cells in alginate beads and Si-coated beads. As expected, we could not obtain any conversion in the biphasic approach in all tested solvents for free Oxd whole cells, namely cyclohexane (C), 2-methyl-tetrahydrofurane (T), methyl-tert-butyl ether (M) and n-hexane (H). In the case of the OxdRE immobilizates, we found conversion in cyclohexane as solvent; however, the conversions were very low and no difference between the coated and the uncoated beads were observed. In the case of OxdB immobilizates we could reach~10% conversion by usage of cyclohexane as solvent for Si-coated beads. Even with the uncoated beads, a conversion of~5% was observed. As we used n-hexane as solvent for TEOS coating, we further investigated n-hexane as solvent and found a conversion of 20% by using Si-coated OxdB immobilizate. This result shows in principle that the beads are more suitable in organic solvents than free whole cells, especially in very unipolar solvents like cyclohexane or n-hexane. This might be caused by a shielding effect of the alginate beads. Generally, we were interested in utilizing the whole-cell immobilizates in an aqueous reaction medium, since we already established a method for the usage of Oxd whole cells in organic medium with a superabsorber. Therefore, we investigated the stability of the beads (calcium alginate beads and Si-coated beads) in buffer containing ethanol as cosolvent. Normally, aldoximes are not very soluble in an aqueous reaction medium, wherefore a cosolvent is needed. In previous studies, it was found that ethanol is a very suitable solvent for the use in combination with Oxd whole-cell catalysts [21]. The activity of free Oxd whole cells and those immobilized with alginate and Si-coated was tested in HEPES buffer with 10% or 50% ethanol as solvent. The free whole cells or the beads were incubated for 24 h in this mixture, and then biotransformations with n-octanaloxime were performed with these pretreated catalysts (Figure 7). We normalized the results on the activity of the free whole cells or immobilized whole cells without incubation.
Catalysts 2020, 10, x FOR PEER REVIEW 13 of 19 shows in principle that the beads are more suitable in organic solvents than free whole cells, especially in very unipolar solvents like cyclohexane or n-hexane. This might be caused by a shielding effect of the alginate beads. Generally, we were interested in utilizing the whole-cell immobilizates in an aqueous reaction medium, since we already established a method for the usage of Oxd whole cells in organic medium with a superabsorber. Therefore, we investigated the stability of the beads (calcium alginate beads and Si-coated beads) in buffer containing ethanol as cosolvent. Normally, aldoximes are not very soluble in an aqueous reaction medium, wherefore a cosolvent is needed. In previous studies, it was found that ethanol is a very suitable solvent for the use in combination with Oxd whole-cell catalysts [21]. The activity of free Oxd whole cells and those immobilized with alginate and Si-coated was tested in HEPES buffer with 10% or 50% ethanol as solvent. The free whole cells or the beads were incubated for 24 h in this mixture, and then biotransformations with noctanaloxime were performed with these pretreated catalysts (Figure 7). We normalized the results on the activity of the free whole cells or immobilized whole cells without incubation.  Relative activity 10% EtOH 50% EtOH Figure 7. Incubation of (a) OxdB and (b) OxdRE whole cells (free, non-immobilized), immobilized in calcium alginate (alginate) or in Si-coated calcium alginate (Si-beads)) in 10% or 50% ethanol in HEPES. The relative activity in comparison with standard activity of dehydration is shown.
We found a very interesting stabilizing effect of the Si-beads in comparison with the free whole cells. While for the whole cells only 20% (OxdB) or ~5% (OxdRE) residual activity was observed after 24 h incubation time in buffer with 10% ethanol, ~80% (OxdB) or ~30% (OxdRE) residual activity was found for the Si-beads. These experiments show that a stabilizing effect of the beads, especially of the coated beads, is obtained. It is very surprising that after the TEOS coating overnight in n-hexane residual activity and an enhanced stability for both of the catalysts (OxdB and OxdRE) is observed, whereas usually only a few minutes of incubation of the whole cells in organic solvents leads to a dramatical loss of activity [22].
Based on these findings, we then performed a recycling study using OxdB whole cells immobilized in Si-coated alginate beads. We did not perform a recycling study for the OxdRE immobilizates because the previous experiments demonstrated higher stability and residual activity of OxdB after immobilization. In our recycling experiment, the conversion of n-octanaloxime to n-octanenitrile in the first round (88%) was normalized to 1, and all experiments with the recycled catalyst were compared to this value (Figure 8). Figure 7. Incubation of (a) OxdB and (b) OxdRE whole cells (free, non-immobilized), immobilized in calcium alginate (alginate) or in Si-coated calcium alginate (Si-beads)) in 10% or 50% ethanol in HEPES. The relative activity in comparison with standard activity of dehydration is shown.
We found a very interesting stabilizing effect of the Si-beads in comparison with the free whole cells. While for the whole cells only 20% (OxdB) or~5% (OxdRE) residual activity was observed after 24 h incubation time in buffer with 10% ethanol,~80% (OxdB) or~30% (OxdRE) residual activity was found for the Si-beads. These experiments show that a stabilizing effect of the beads, especially of the coated beads, is obtained. It is very surprising that after the TEOS coating overnight in n-hexane residual activity and an enhanced stability for both of the catalysts (OxdB and OxdRE) is observed, whereas usually only a few minutes of incubation of the whole cells in organic solvents leads to a dramatical loss of activity [22].
Based on these findings, we then performed a recycling study using OxdB whole cells immobilized in Si-coated alginate beads. We did not perform a recycling study for the OxdRE immobilizates because the previous experiments demonstrated higher stability and residual activity of OxdB after immobilization. In our recycling experiment, the conversion of n-octanaloxime to n-octanenitrile in the first round (88%) was normalized to 1, and all experiments with the recycled catalyst were compared to this value ( Figure 8). Figure 8, a loss of activity during the recycling study is observed; however, within the first three recycling steps, more than 85% residual activity is obtained. After five rounds of recycling, only ~20% residual activity of the catalyst is left.

Discussion
Different immobilization techniques of Oxds were tested using purified Oxds and Oxds in whole cells. We focused on two enzymes, namely OxdB from Bacillus sp. OxB-1 and OxdRE from Rhodococcus erythropolis. Both are active for the dehydration of n-octanaloxime to n-octanenitrile, which was used as a standard substrate in every activity assay. First, we investigated different immobilization carriers, which immobilize free enzymes, by hydrophobic interactions or covalently. We chose three different hydrophobic carriers (h1, h2 and h3), one amino carrier (a1) which is preactivated by glutaric aldehyde; and two epoxy carriers (e1 and e2). It was found that the residual activity after immobilization with high immobilization efficiencies (~80-90%) was <20% in all cases. The strong loss of activity for all carriers is probably due to the instability of the purified enzyme at elevated temperatures. We could show that even an incubation of the purified enzymes for 1 h at 30 °C in buffer leads to a loss of activity of 50%. During immobilization, the enzymes are shaken for 24 h hours at temperatures above room temperature (rt) (see Supporting Information), which partly explains the severe loss of activity. Although the remaining activities after immobilization of the free enzymes were very low, an additional cosolvent screening was performed to determine the stability of the immobilizates in comparison with free purified enzymes. The immobilization of the enzymes seems to have a small stabilizing effect with and without the co-solvent. These results led us to the conclusion that purified Oxds are not very stable and that immobilization of the enzymes has a positive effect on the stability, while the activity is drastically decreased. However, the strong decrease in activity during immobilization makes it difficult to use in organic chemistry, and we decided to switch our investigations to the immobilization of whole cells, since it was already found that Oxds are more stable in the whole cells compared to the purified enzymes [27]. Using an As shown in Figure 8, a loss of activity during the recycling study is observed; however, within the first three recycling steps, more than 85% residual activity is obtained. After five rounds of recycling, only~20% residual activity of the catalyst is left.

Discussion
Different immobilization techniques of Oxds were tested using purified Oxds and Oxds in whole cells. We focused on two enzymes, namely OxdB from Bacillus sp. OxB-1 and OxdRE from Rhodococcus erythropolis. Both are active for the dehydration of n-octanaloxime to n-octanenitrile, which was used as a standard substrate in every activity assay. First, we investigated different immobilization carriers, which immobilize free enzymes, by hydrophobic interactions or covalently. We chose three different hydrophobic carriers (h1, h2 and h3), one amino carrier (a1) which is preactivated by glutaric aldehyde; and two epoxy carriers (e1 and e2). It was found that the residual activity after immobilization with high immobilization efficiencies (~80-90%) was <20% in all cases. The strong loss of activity for all carriers is probably due to the instability of the purified enzyme at elevated temperatures. We could show that even an incubation of the purified enzymes for 1 h at 30 • C in buffer leads to a loss of activity of 50%. During immobilization, the enzymes are shaken for 24 h hours at temperatures above room temperature (rt) (see Supporting Information), which partly explains the severe loss of activity. Although the remaining activities after immobilization of the free enzymes were very low, an additional cosolvent screening was performed to determine the stability of the immobilizates in comparison with free purified enzymes. The immobilization of the enzymes seems to have a small stabilizing effect with and without the co-solvent. These results led us to the conclusion that purified Oxds are not very stable and that immobilization of the enzymes has a positive effect on the stability, while the activity is drastically decreased. However, the strong decrease in activity during immobilization makes it difficult to use in organic chemistry, and we decided to switch our investigations to the immobilization of whole cells, since it was already found that Oxds are more stable in the whole cells compared to the purified enzymes [27]. Using an immobilization technique and comparing the use of calcium alginate beads to calcium alginate beads coated with TEOS, we successfully immobilized OxdB and OxdRE in whole cells with residual activities of up to~70%. Compared to the immobilization of purified enzymes, this residual activity is remarkable. For both enzymes, we could observe higher activities for the Si-coated beads in comparison with the uncoated beads, which is probably due to a better diffusion of the substrate in the beads. The alginate beads have a very hydrophilic surface, which makes diffusion of the hydrophobic substrate n-octanaloxime difficult. The Si-coating seems to mask the hydrophilic surface and make them more hydrophobic, which makes diffusion easier. Since TEOS coating to gain Si-coated alginate beads is performed in n-hexane as solvent, we performed reactions in pure organic solvents using immobilized cells compared to free cells. Conversion was found only in the case of OxdB in n-hexane or cyclohexane as solvent, leading us to the conclusion that the alginate beads are not suitable for use in organic solvents. Superabsorber immobilization of Oxd whole cells was already successfully established for use in organic solvents; thus, we did not further investigate the use of alginate beads in organic media [22]. It was found that the alginate beads, especially the Si-coated beads, are significantly more stable in ethanol-containing buffer with~80% residual activity after 24 h incubation time in 10% ethanol of OxdB in Si-coated beads compared to~20% for the free cells. Recycling of the beads led to decreased residual activity; however, the immobilizates can be used three times before a decrease in the activity of <85% is observed.