Characterization of different biocatalyst formats for BVMO‐catalyzed cyclohexanone oxidation

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer–Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax‐derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Furthermore, we determined and compared the cyclohexanone conversion kinetics for different CHMO‐biocatalyst formats, that is, isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO‐containing P. taiwanensis VLB120. Biofilms showed less favorable values for KS (9.3‐fold higher) and kcat (4.8‐fold lower) compared with corresponding KM and kcat values of isolated CHMO, but a favorable KI for cyclohexanone (5.3‐fold higher). The unfavorable KS and kcat values are related to mass transfer‐ and possibly heterogeneity issues and deserve further investigation and engineering, to exploit the high potential of biofilms regarding process stability. Suspended cells showed only 1.8‐fold higher KS, but 1.3‐ and 4.2‐fold higher kcat and KI values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

utilize O 2 as oxygen donor, and depend on NAD(P)H (Ryerson et al., 1982). They feature high regio-, stereo-, and enantioselectivities and operate under mild reaction conditions, making them an environmentally friendly alternative to the existing chemical catalytic processes (Ten Brink et al., 2004).
One of the main features of BVMOs is their broad substrate scope, also covering nonnatural substrates. Besides the carbonyl carbon in aliphatic, cyclic, and aromatic ketones, BVMOs also oxidize sulfur (Colonna et al., 1996), nitrogen (Ottolina et al., 1999), and even selenium (Latham et al., 1986) atoms. In the last two decades, extensive work has been done regarding the isolation of BVMOs and their evaluation for the generation of novel functionalities with value for the pharmaceutical, food, and fine chemical industries (Alphand et al., 2003;Fürst et al., 2019;Pazmino et al., 2010). On the downside, most BVMO-based oxidation processes suffer from low enzyme stability and inhibitory or toxic effects of substrates and/or products restricting volumetric productivities and product titers (Furst et al., 2019).
The application of BVMOs in in vivo and/or immobilized formats constitutes a promising strategy to improve biocatalyst stability and total turnover number. However, a change in biocatalyst configuration can affect reaction kinetics and, consequently, reaction performance . Typically, in vitro kinetics are characterized under conditions that do not resemble in vivo environments, and thus reaction kinetics often differ among in vitro and in vivo formats (Teusink et al., 2000;Van Eunen & Bakker, 2014).
Conversely, other studies that characterized in vivo catalytic rates found that they generally concur with in vitro measurements (Davidi et al., 2016;Heckmann et al., 2020). Such contradictory results also have been reported for the comparison of kinetics for suspended and immobilized microbial cells. Whereas toluene degradation kinetics were comparable in biofilms and planktonic cells (Mirpuri et al., 1997), nitriloacetate degradation activity was three-fold enhanced for sand-associated as compared with suspended cells (McFeters et al., 1990). These findings imply that similarity or differences in reaction kinetics among biocatalyst formats might be case-dependent, and point out that the determination and understanding of differences in kinetics is of significant interest for modeling biological systems and selecting the most promising biocatalyst format for technical applications.
In the present work, we aimed to understand if, to what extent, and why CHMO-reaction kinetics concur or differ among isolated enzyme-, suspended cell-, and biofilm-based formats. For this purpose, CHMO from Acidovorax sp. CHX100 was introduced into Pseudomonas taiwanensis VLB120, a solvent-tolerant strain and good biofilm former (Halan et al., 2011;Rohan Karande et al., 2014;Volmer et al., 2014). This strain was used for recombinant CHMO production and as a catalytic unit in suspended cell-and biofilm formats.

| Construction of the phylogenetic tree
Amino acid sequences of different BVMOs were aligned using the MUSCLE algorithm (Edgar, 2004). The evolutionary history was inferred by using the Maximum Likelihood method and the Whelan and Goldman model (Whelan & Goldman, 2001). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with a superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 21,745]). This analysis involved 36 amino acid sequences. There were a total of 788 positions in the final data set.
Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

| Chemicals, media, and bacterial strains
Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich or Carl Roth in the highest purity available and used without further purification. Microbial strains and plasmids used in this study are listed in Table 1. Cells were grown in lysogeny broth (LB) medium T A B L E 1 Strains and plasmids used in this study

| Purification protocol
Cells were resuspended in 100 mM Kpi buffer (pH = 7.4) to an OD 450 of 50 and disrupted by using a French press (Thermo Electron Corporation). The sample was passed three times at 1200 psi. The crude cell extract was centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was loaded on a disposable plastic column (Thermo Fisher Scientific), which was packed with Strep-Tactin ® Superflow ® resin (IBA Life Sciences) and equilibrated following the manufacturer's instructions. The flowthrough, wash, and elution fractions were collected for SDS-PAGE analysis. In total three elution fractions were collected (1.5 ml, 2 ml, and 1.5 ml). The column was regenerated and stored in the wash buffer at 4°C until reuse.

| Determination of CHMO activity
To evaluate purification efficiency and the substrate spectrum of CHMO, its activity was assayed by monitoring the decrease in NADPH absorbance at 340 nm after the addition of substrate with a Cary Bio 300 UV-visible spectrophotometer (Varian, Palo Alto, USA). Activity assays were performed at 30°C for at least 2 min.
Assay mixtures contained 1 mM substrate, 0.2 mM NADPH, and 20 µl enzyme solution (containing 1.0-2.7 mg CHMO ml −1 ) in 1 ml total volume. Initial activities were calculated from the decrease of NADPH absorption at 340 nm for 60-120 s using a specific absorption coefficient of ε = 6.22 mM −1 cm −1 . One unit of enzyme activity was defined as 1 µmol of NADPH consumed per min.

| Determination of CHMO kinetics
For kinetic analyses, a cell concentration of 0.25 g CDW L −1 or 20 µl of purified enzyme (0.98 mg CHMO ml −1 ) were used in 100 mM potassium phosphate buffer, pH = 7.4 (Kpi buffer) supplemented with 1% (w/v) glucose for whole cells as catalysts. For the variation of cyclohexanone and NADPH concentrations, the assays were conducted in 2 ml Eppendorf reaction tubes on a thermoshaker (Thermomixer C, Eppendorf). For the variation of the O 2 concentration, small glass vials with a gas-tight septum cap were used. Buffercontaining vials were incubated at 60°C for 10 min and then degassed with N 2 for 45 s. Then, target amounts of O 2 were added with a gas-tight syringe (Hamilton). The assay was started by the addition of cyclohexanone for whole cells, and of cyclohexanone and CHMO for the isolated enzyme. Reactions were carried out for 5 min and stopped by the addition of ice-cold diethyl ether containing 0.2 mM n-decane as an internal standard. After 2 min of extraction by vortexing and centrifugation, the organic phase was dried over waterfree Na 2 SO 4 before it was transferred to a GC vial for analysis.

| Determination of CHMO kinetics in biofilms
The biofilm capillary reactor system and P. taiwanensis VLB120 (pSEVA_CHMO) pre-cultures were prepared as reported before . A serological pipette functioned as a capillary for biofilm growth (3 mm inner diameter, 10 cm length, Labsolute, Th. Geyer GmbH & Co. KG). M9* medium (5 g L −1 glucose) was supplied using a peristaltic pump (530S with 205CA12 pump head, Watson-Marlow). The capillaries of the reactor system were inoculated by purging 2 ml M9* pre-culture through the injection port.
The medium flow was started 2 h after inoculation at a rate of 150 μl min −1 . Air segments were introduced 2 days after inoculation at a rate of 150 μl min −1 . The airflow rate was set to 200 µl min −1 4 days after inoculation and increased to 400 µl min −1 at Day 5. By the addition of 1 mM IPTG to the medium feed, heterologous expression of BVMO genes was induced on Day 5. Bubble traps, as well as sampling ports, were attached at the end of the capillary to enable gas and liquid sampling while injection ports were removed from the setup. The kinetics experiment was conducted on Day 6. The airflow rate was set to 600 µl min −1 and feed solutions containing desired cyclohexanone concentrations were freshly prepared in separate medium bottles (Kpi buffer, pH 7.4, 10 g L −1 glucose, 1 mM IPTG).
The desired cyclohexanone feed solution was supplied to the capillaries by using PTFE tubing and a peristaltic pump (Tygon MHLL pump tubing, IPC 4, Ismatec) at a flow rate of 150 µl min −1 equaling a residence time of 5 min. Thirty-minute after the switch to cyclohexanone containing feed, a sample was collected for 15 min and directly prepared for GC (as described before) and HPLC analysis.
The HPLC sample was centrifuged (10 min, 4°C, 17,000 × g). One hundred microliters of the supernatant was acidified with 10 µl 1 M HCl and subjected to HPLC analysis. The procedure was repeated BRETSCHNEIDER ET AL.

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with the different feed solutions. Finally, the biomass was harvested from the capillary and dried for 5 days at 80°C for cell dry weight determination.

| Analytical methods
Biomass concentrations were detected as the optical density at a wavelength of 450 nm (OD 450 ) using a Libra S11 spectrophotometer (Biochrom). One OD 450 unit corresponds to 0.186 g CDW L −1 (Halan et al., 2010).
Protein concentrations were determined using BSA as protein standard (Quick StartTMBradford Protein Assay) following the supplier's instructions. Expression patterns were analyzed via SDS-PAGE according to Laemmli (1970). CHMO was quantified by determining the integrated density of CHMO bands using ImageJ.
Samples with known CHMO content were used as calibration standards to calculate the CHMO content within samples ( Figure S3).
The kinetic parameters V max , K M (or K S ), and K I were calculated in Matlab 6.1 and fitted to the following equations using the method of least squares: (1) without substrate inhibition (O 2 as limiting substrate) (2) with substrate inhibition (NADPH or cyclohexanone as lim- 3 | RESULTS

| CHMO gene expression in and isolation from P. taiwanensis VLB120
In our previous studies, the CHMO gene of Acidovorax sp. CHX100 was isolated and applied within an in vivo cascade to produce ε-caprolactone (ε-CL), 6-hydroxycaproic acid, and 6-aminocaproic acid from cyclohexane in P. taiwanensis VLB120 and E. coli R. Karande et al., 2017;. Solvent-tolerant P. taiwanensis VLB120 is a good biofilm former and has been intensively studied regarding whole-cell redox biocatalysis (Lang et al., 2014;Volmer et al., 2014;Wynands et al., 2018). In the present work, this strain was selected for CHMO gene expression and enzyme isolation and as the host strain for kinetic studies on suspended cells and biofilms. Recombinant CHMO was isolated from P. taiwanensis VLB120 crude-cell extracts via a one-step protocol using a Strep-tactin resin resulting in a purification factor of 8.5 and a CHMO activity of 0.94 U mg CHMO −1 (Table 2, Figure S1). The CHMO protein in the eluted fraction leads to a light yellow-colored solution due to the tightly bound FAD cofactor (Fraaije et al., 2005). The absorbance spectrum of CHMO showed the two maxima at 380 and 443 nm characteristic for flavins and flavoproteins ( Figure S2).

| Substrate spectrum and catalytic performance of CHMO
BVMOs are known to display a wide range of substrate spectra covering over 100 compounds (Mihovilovic et al., 2002). To get a rough overview on the substrate spectrum of the Acidovorax CHMO, activities were analyzed spectrophotometrically, that is, in terms of NADPH consumption (1 mM substrate, 2-5 min reaction time), as done in previous studies Brzostowicz et al., 2003;Trower et al., 1989). With a focus on initial activities, we investigated a large variety of substrates, including cyclic, substituted cyclic, aromatic, and alkylic ketones, as well as thioanisole, its p-methoxy derivative, and methyl phenyl sulfoxide.
Enantio-and regioselectivities were not investigated. Chiral substrates were applied as racemates.
As expected from the involvement of CHMO in cyclohexane degradation (Salamanca & Engesser, 2014), cyclohexanone was among the best-converted substrates (Figure 3). CHMO did not show any uncoupling, neither with cyclohexanone as substrate nor without substrate (Supporting Information Section 1, Table S1). Similarly, uncoupling may not be prominent for other substrates, but cannot be excluded. The highest activity was found for 3-methylcyclohexanone.
The position of methyl substitutions of cyclohexanone strongly influenced CHMO activity with 54%, 104%, and 91% relative activity for methyl groups at positions 2, 3, and 4, respectively. Whereas the bulky substrate 3,3,5-trimethylcyclohexanone was converted with 85% relative activity, 4-tert-butylcyclohexanone reacted more slowly (12% relative activity). Alkylic ketones also were converted, but at lower rates than cyclic compounds. Even lower rates were found for benzylic ketones, whereas substrates with the carbonyl group further away from aromatic rings as, for example, β-tetralone (65% relative activity), were more preferred. BVMOs are well known to catalyze sulfide and sulfoxide oxidations (Bisagni, Summers, et al., 2014;Colonna et al., 1998;Zhang et al., 2018), which also was confirmed here for Acidovorax CHMO. Overall, CHMO showed a large substrate spectrum with high activities towards cyclic compounds with or without substitutions as well as for sulfides and sulfoxides and lower activities towards aliphatic and benyzlic ketones.
The biocatalytic performance of isolated CHMO was further characterized in biotransformations conducted for 6 h with 5 mM of seven substrates from four different compound classes. All products except δ-valerolactone, which could not be detected with the chromatographic method, were subjected to GC-MS analysis confirming their structure (Figures S4-S9). The lower conversions for β-tetralone (24%) and methylphenylsulfoxide (57%) (Figure 4)  with these substrates might be prominent leading to the formation of reactive oxygen species (ROS) and thus enzyme destabilization. The high conversions obtained for (substituted) cycloalkanes qualify them as preferred substrates of Acidovorax-CHMO (Figure 4).

| In vitro characterization of CHMO kinetics
CHMO in vitro kinetics for cyclohexanone conversion was investigated by varying either the cyclohexanone, NADPH, or O 2 concentration and measuring initial reaction rates based on ε-CL formation. These rates followed Michaelis-Menten kinetics with substrate inhibition for cyclohexanone and NADPH ( Figure 5).
Parameters were fitted utilizing the respective equations (Table 3).

| Characterization of CHMO kinetics in suspended cell-and biofilm formats
To compare biocatalyst formats, we aimed to estimate the kinetic parameters for suspended cells and biofilms. Cyclohexane, as well as O 2 concentrations, were varied to analyze the respective kinetics of T A B L E 3 Cyclohexanone monooxygenase (CHMO) kinetics for isolated enzyme-, suspended cell-, and biofilm-based biocatalyst formats  (Table 3). Similarly, the cyclohexanone-related substrate inhibition constants (K I ) of suspended cells and biofilms were 4.3and 5.3-fold higher than those of isolated CHMO. These results indicate more prominent cyclohexanone and O 2 mass transfer limitations towards and into cells and, especially, biofilms.
The maximal specific activity of suspended cells (V max ) was almost 10-fold higher than that of the biofilm (Table 3). To further characterize this effect, CHMO-related k cat values for both in vivo formats were estimated based on CHMO contents of respective biomass. The latter were derived by relating SDS-PAGE band intensities to those obtained with samples of known CHMO content ( Figure S3) and assuming a total protein fraction of 55% in cell dry mass (Neidhardt et al., 1990). Remarkably, the obtained k cat value for CHMO in suspended cells and cyclohexanone as substrate was 1.3-fold higher than that obtained for isolated CHMO (Table 3). In biofilms, however, this k cat value was 4.8-fold lower than isolated CHMO. This result indicates that the CHMO content, as well as active enzyme fractions, were lower in biofilms than in suspended cells.
Overall, the significant differences in kinetic parameters obtained for the different CHMO biocatalyst formats emphasize that the biocatalyst format choice plays an important role regarding biocatalyst and thus bioprocess efficiency.

| Characteristics of Acidovorax CHMO
In this study, a Type I Baeyer-Villiger monooxygenase involved in cyclohexane degradation by Acidovorax sp. CHX100 was isolated and characterized (Salamanca & Engesser, 2014). The Acidovorax CHMO was shown to integrate well into the clustering of BVMO gene sequences according to their native substrate (Fraaije et al., 2002) ( Figure 1). CHMO gene expression in P. taiwanensis VLB120 by means of the pSEVA244_T vector resulted in high expression levels of the soluble protein ( Figure 2) with a minor effect on growth, qualifying P. taiwanensis VLB120 as suitable host for CHMO synthesis. Acidovorax CHMO showed a large substrate spectrum as it is quite common for BVMOs Brzostowicz et al., 2003;Riebel et al., 2012), also catalyzing sulfur oxidation in substrates that are structurally different from their native substrate (Fink et al., 2012;Fraaije et al., 2005). For cyclic and substituted cyclic substrates, Acidovorax CHMO enabled 90%-100% conversion within 6 h of reaction ( Figure 4). As observed for other CHMOs, its activity towards benzylic ketones such as acetophenone and α-tetralone was very low (Riebel et al., 2012). The most studied BVMO from Acinetobacter has been shown to accept over 100 different substrates (Mihovilovic et al., 2002;J. D. Stewart, 1998).
Other BVMOs like the phenylacetone monooxygenase from T. fusca have a more restricted substrate spectrum (Fraaije et al., 2005). The substrate screen given in this study revealed a versatile CHMO, of which the substrate spectrum deserves further investigation, including the determination of enantio-and regiospecificities.
The turnover numbers (k cat ) of Acidovorax CHMO and its K m value for the native substrate cyclohexanone are within the typical ranges reported for BVMOs (Table 4). It has to be noted that studies on BVMO kinetics often rely on spectrophotometrically analyzed NADPH oxidation and can be compromised by a possible uncoupling leading to overestimated activities. Whereas Acidovorax CHMO did not show uncoupling with cyclohexanone as substrate, which is in contrast to other BVMOs, for example, those originating from T. municipale or Gordonia sp. showing 11% and 19% uncoupling,   Donoghue et al., (1976); Kamerbeek et al. (2004) Thermocrispum municipale 4-Methyl-cyclo-hexanone 1,400 0.11 ; Kamerbeek, Olsthoorn, et al. (2003) Pseudomonas putida JD1 respectively (Fordwour et al., 2018;Li et al., 2017). Whereas low K Mvalues for NADPH (1-64 µM) have been reported for other BVMOs (Table 4), Acidovorax CHMO exhibited a comparably high K M (372 µM), which is still within the intracellular range of 120-540 µM as determined for E. coli (Bennett et al., 2009;Milo et al., 2010, BNID:100146), but indicates a firm dependency of whole-cell-based CHMO-catalysis on the cellular redox state.
Substrate inhibition is a well-known phenomenon for CHMOs (Alphand et al., 2003;Delgove et al., 2018;Hilker et al., 2008) and also was found for Acidovorax CHMO with a K I of 2.24 mM. For synthetic application, this demands suitable substrate feeding strategies. For CHMO Acinetobacter and PAMO T.fusca , the product NADP + has moreover been found to act as a competitive inhibitor (K I = 38 and 2.7 µM, respectively) (Ryerson et al., 1982;Torres Pazmiño et al., 2008). Whereas such product inhibition was not found for Acidovorax CHMO, substrate inhibition by NADPH was apparent, which has been reported for BVMOs so far. The respective K I (1.85 mM), however, was clearly above typically encountered intracellular NADPH concentrations, for example,120-540 µM in E. coli (Bennett et al., 2009;Milo et al., 2010, BNID:100146).
The K M of 1.1 µM obtained in this study translates to a catalytic efficiency of 10,800 s −1 M −1 , which is in line with these previous studies.

| Kinetic parameters differ for different biocatalyst formats
In recent years, in vivo kinetic parameters and their correspondence to in vitro counterparts have been questioned and refined using omics approaches (Davidi et al., 2016;Heckmann et al., 2020 (Bennett et al., 2009;Milo et al., 2010, BNID:100146).
The higher k cat in suspended cells can be explained by the intracellular milieu, for which enzymes are evolutionarily optimized (Cheung et al., 2005). Such conditions are difficult to realize with standard reaction buffers. Furthermore, partial enzyme denaturation during purification can affect the in vitro k cat estimation. These differences among in vivo and in vitro kinetics (Table 3) can bring advantages for in vivo biocatalysis. Besides an optimal milieu enabling high enzyme stability and effective metabolism-based redox cofactor regeneration, continuous enzyme regeneration/synthesis constitutes another advantage of in vivo biocatalysis (Kadisch et al., 2017;Schrewe et al., 2013).
In biofilms, self-immobilized cells are embedded within a selfproduced matrix of extra-polymeric substances (EPS). Compared with suspended cells, apparent K S and K I values of biofilms for cyclohexanone were 5.2-and 1.3 times higher, respectively, and the V max was 9.6-fold lower, which only partially was attributed to a lower BVMO content (the k cat was 6.3 time lower, Table 3). Possible reasons for these differences include the substrate mass transfer within a biofilm, which mainly depends on diffusion resulting in concentration gradients and consequently a higher apparent K S . Further, the high heterogeneity among cells within biofilms (P. S. Stewart & Franklin, 2008;Wimpenny et al., 2000) imply that not all cells are catalytically active, resulting in a reduced V max .
However, planktonic cell-based kinetics are often used to model biofilmbased processes (Bakke et al., 1984;Mirpuri et al., 1997), which, as exemplified by the results obtained in this study, can lead to a substantial overestimation of biological activity.
Apart from reaction kinetics and thus the biotransformation rate, the stability of biocatalyst formats is an important parameter, as it determines the product yield on biocatalyst (g product g catalyst −1 ) and the achievable product titer Hoschek, Toepel, et al., 2019;Kadisch et al., 2017). Thus, it will be the task of future research on the process performance of different biocatalyst formats to focus on stability aspects and combine them with rate-and specificity-related assessments (Tufvesson et al., 2011).

| CONCLUSIONS
A BVMO originating from Acidovorax CHX100 was heterologously expressed in P. taiwanensis and characterized in the isolated form.
Like other BVMOs, this enzyme was found to feature a broad substrate spectrum and showed the highest activity towards BRETSCHNEIDER ET AL.
| 2729 cyclic ketones. Unlike other CHMOs, no uncoupling was observed with and without cyclohexanone as substrate. Kinetics was also found to be similar as reported for other CHMOs and was characterized in detail, not only for the isolated enzyme but also for CHMO-containing suspended cells and biofilms to compare different biocatalyst formats. This kinetic assessment revealed slightly higher K S and k cat values for suspended cells compared with the K M and k cat of the isolated enzyme. Biofilms exhibited the lowest k cat and the highest K s . Both suspended cells and biofilms were significantly less susceptible to inhibition by cyclohexanone than isolated CHMO.
From a kinetics point of view, the suspended-cell format can thus be considered most promising, as it efficiently exploits the enzyme capacity and NADPH regeneration via glucose metabolism. The biofilm format bears high potential regarding process stability but suffers from kinetic issues related to mass transfer and possibly heterogeneity, which deserve further research and engineering efforts.

ACKNOWLEDGMENTS
We acknowledge the use of the facilities of the Centre for Biocatalysis

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.