This is the submitted version of the following article: Delgore, M.A.F., et al. High performing immobilized Baeyer-Williger monooxygenase and glucose dehydrogenase for the synthesis of Ɛ-caprolactone derivate in Applied catalysis A (Ed. Elsevier), vol. 572 (Feb. 2019), p. 134-141. Which has been published in final form at DOI 10.1016/j.apcata.2018.12.036. © 2019. This manuscript version is made available under the CC-BY- NC-ND 4.0 license http://creativecommons.org/licenses/by-nc- nd/4.0/ 1 High performing immobilized Baeyer-Villiger monooxygenase and glucose 2 dehydrogenase for the synthesis of Ɛ-caprolactone derivative 3 Marie A. F. Delgovea, Daniela Valenciab, Jordi Soléb, Katrien V. Bernaertsa, Stefaan M. 4 A. De Wildemana, Marina Guillénb, Gregorio Álvarob a 5 Maastricht University, Aachen-Maastricht Institute for Biobased Materials (AMIBM), 6 Brightlands Chemelot campus, Urmonderbaan 22, 6167 RD Geleen, The Netherlands b 7 Bioprocess Engineering and Applied Biocatalysis Group, Department of Chemical, 8 Biological and Environmental Engineering, Universitat Autònoma de Barcelona, 08193 9 Bellaterra, Catalonia, Spain 10 Corresponding author: 11 Gregorio Álvaro 12 Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de 13 Barcelona, 08193 Bellaterra, Catalonia, Spain 14 Telephone: +34 93 581 2791 15 Fax: +34 93 581 2013 16 gregorio.alvaro@uab.cat 17 Authors’ e-mail addresses 18 Marie A. F. Delgovea: marie.delgove@maastrichtuniversity.nl; Daniela Valencia: 19 danielapatricia.valencia@uab.cat; Jordi Soléb: jordi.jole.jerre@uab.cat; Katrien V. Bernaertsa: 20 katrien.bernaerts@maastrichtuniversity.nl; Stefaan M. A. De Wildemana: 21 s.dewildeman@maastrichtuniversity.nl; Marina Guillénb: marina.guillen@uab.cat 1 22 ABSTRACT 23 The industrial application of Baeyer-Villiger monooxygenases is typically hindered by stability 24 and cofactor regeneration considerations. Stability of biocatalysts can be improved by 25 immobilization. The goal of this study was to evaluate the (co)-immobilization of a 26 thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) 27 with a glucose dehydrogenase (GDH) from Thermoplasma acidophilum for NADPH cofactor 28 regeneration. 29 Both enzymes were immobilized on an amino-functionalized agarose-based support (MANA- 30 agarose). They were applied to the synthesis of 3,3,5-trimethylcyclohexanone for the synthesis 31 of ε-caprolactone derivatives which are precursors of polyesters. The performances of the 32 immobilized biocatalysts were evaluated in reutilization reactions with up to 15 cycles and 33 compared to the corresponding soluble enzymes. Co-immobilization proved to provide the 34 most efficient biocatalyst with an average conversion of 83% over 15 reutilization cycles 35 leading to a 50-fold increase of the biocatalyst yield compared to the use of soluble enzymes 36 which were applied in a fed-batch strategy. 37 TmCHMO was immobilized for the first time, with very good retention of the activity 38 throughout reutilization cycles. This immobilized biocatalyst contributes to the application of 39 BVMOs in up-scaled biooxidation processes. 40 Keywords: biocatalyst immobilization, Baeyer-Villiger monooxygenase, lactone monomer, 41 cofactor recycling, glucose dehydrogenase 42 43 2 44 1. INTRODUCTION 45 Enzymatic reactions have been identified as a sustainable technology since they usually follow 46 the rules of green chemistry.[1,2] Oxidative biocatalysis, and Baeyer-Villiger monooxygenases 47 (BVMOs) in particular, is an alternative of lesser toxicity compared to chemical oxidation.[3] 48 BVMOs are biocatalysts capable of catalyzing the oxidation of (cyclic) ketones by inserting 49 one atom of oxygen in a C-C bond, therefore generating water as by-product. BVMOs can 50 catalyze the oxidation of a wide range of cyclic ketones of various ring size including alkyl 51 substituted ketones, as well as perform enantioselective sulfoxidation.[4-6] These oxidative 52 enzymes have been applied to the synthesis of intermediates for the pharmaceutical industry,[7- 53 9] and chiral molecules for fine chemical and fragrances.[10] Additionally, several BVMOs 54 have been identified as relevant biocatalyst for the synthesis of lactone as monomers for 55 polymeric materials, for example, ε-caprolactone, either from whole-cell[11] or via a cascade 56 reaction,[12] lauryl lactone,[13] a nitrile-substituted ε-caprolactone as precursor for 57 polyamide,[14] and β,δ-trimethyl-ε-caprolactone (TMCL).[15,16] Alkyl substituted lactones 58 are particularly interesting for the synthesis of polyesters with low glass transition temperature 59 (Tg < 0 °C in general).[17] This property enables applications such as biodegradable 60 plasticizers[18] or encapsulating agents for coating formulations[19] with polymers from 61 TMCL for example. 62 The applicability of BVMOs is however hindered by their lack of robustness, either due to 63 thermostability or to limited stability in the presence of organic solvents. Using protein 64 engineering, several mutants of cyclohexanone monooxygenase with improved thermostability 65 were created.[20-22] The discovery of new thermostable BVMOs contributes to the 66 development of their applicability in biotransformations.[23-26] Recently, a cyclohexanone 67 monooxygenase from Thermocrispum municipale DSM 44069 (TmCHMO; EC 1.14.13.22) 3 68 was identified as being particularly relevant for the preparation of lactones as polymeric 69 building blocks due to its high thermostability, good resistance to organic solvents, and broad 70 substrate scope towards cyclic ketones.[27,28] 71 Although TmCHMO has already been applied for the synthesis of ε-caprolactone derivatives 72 from 3,3,5-trimethylcyclohexane, using either a self-sufficient fused biocatalyst[15] or a 73 glucose dehydrogenase to regenerate the NADPH co-factor,[16] this enzyme has not yet been 74 immobilized. Immobilization of whole-cells or isolated enzymes is indeed known to increase 75 the operational stability of enzymes. Additionally, immobilization has several advantages 76 including facilitating the recovery of the biocatalyst, decreasing the costs of downstream 77 processing, and potentially decreasing the enzyme cost per kilogram of product, provided that 78 the immobilized biocatalysts maintain their activity throughout the reuses.[29,30] 79 So far, isolated BVMOs have mostly been immobilized to polymeric supports by covalent 80 binding.[31] For example, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus 81 (AcCHMO) was immobilized on Eupergit (polyacrylamide based supported beads) via 82 covalent binding with a glucose 6-phosphate dehydrogenase for the synthesis of chiral lactone 83 building blocks.[32] Fusions of AcCHMO with a polyol dehydrogenase were similarly 84 immobilized for the synthesis of ε-caprolactone.[33] The immobilized biocatalyst, however, 85 displayed a low stability on the support and a poor operational stability. Recently, MANA- 86 agarose (monoaminoethyl-N-aminoethyl)-agarose was identified as a suitable support for the 87 immobilization of a fused AcCHMO-phosphite dehydrogenase (AcCHMO-PTDH).[34] For 88 this enzyme, a higher retained activity was achieved with metal-chelate supports such as Ni- 89 iminodiacetic acid (Ni-IDA) and Co-IDA.[34] 90 In this article, our goal is to expand the use of immobilized BVMOs and evaluate them for the 91 synthesis of lactones as polymeric building blocks. The immobilization of TmCHMO and a 4 92 glucose dehydrogenase from Thermoplasma acidophilum (EC 1.1.1.47) (GDH-Tac) are 93 described with the aim of oxidizing 3,3,5-trimethylcyclohexanone to alkyl substituted ε- 94 caprolactone derivatives (Figure 1). The enzymes were immobilized on a MANA-agarose 95 support, either separately or co-immobilized on the same support, by covalent bonding. The 96 performances of the immobilized enzymes were evaluated in over 15 repeated biooxidation 97 cycles and compared to the corresponding soluble enzymes. 98 2. EXPERIMENTAL 99 2.1.Chemicals. 100 3,3,5-Trimethylcyclohexanone (98%, Sigma-Aldrich), methanol (Biosolve), (+)-glucose 101 (>99%, Alfa Aesar) were used as received. High-density aminoethyl 4BCL agarose (MANA- 102 agarose, Agarose Beads Technologies) was stored at 4 ˚C. β-Nicotinamide adenine 103 dinucleotide phosphate disodium salt (NADP+, 97%, Alfa Aesar), and N-(3- 104 dimethylaminopropyl)-N′-ethylcarbodiimide (EDC, ≥97%, Sigma-Aldrich) were stored at - 105 20 ˚C. TmCHMO and GDH-Tac were produced and supplied by InnoSyn BV (Geleen, The 106 Netherlands). 107 2.2.TmCHMO and GDH-Tac activity assays 108 TmCHMO activity in the CFE was determined spectrophotometrically following NAPDH 109 consumption at 340 nm (Ɛ = 6.22 mM-1 cm-1) with cyclohexanone as a substrate. The mixture 110 contained Tris-HCl (50 mM, pH 8.5), cyclohexanone (0.5 mM), NADPH (0.1 mM). One unit 111 of TmCHMO (U) was defined as the amount of enzyme required to catalyze the conversion of 112 1 µmol of NADPH to NADP+ per min at 20 °C and pH 8.5.[34] 113 GDH-Tac activity was determined spectrophotometrically at 340 nm following the NADP+ (Ɛ 114 = 6.22 mM-1 cm-1, 400 µM) consumption using D-Glucose (200 mM) as substrate and sodium 115 phosphate buffer 100 mM pH 8.0.[35] The basal production of NADPH by unspecific enzymes 5 116 present in the lysate was determined by this same test but avoiding the addition of substrate 117 and adding buffer instead. This production rate is subtracted from the measurement with D- 118 glucose. One unit of activity (U) was defined as the enzyme required to convert 1 µmol of 119 NADP+ per minute at those given conditions (30 ºC, pH 8.0). The absorbance was recorded 120 using a spectrophotometer Cary 50 Bio UV-visible (Palo Alto, USA). 121 2.3.Preparation of immobilized TmCHMO and immobilized GDH-Tac 122 The general procedure for the covalent immobilization of the enzymes on MANA-agarose 123 (density: 1.07 g mL-1) comprised three main steps: i) the ionic adsorption of the enzyme to the 124 support, ii) the addition of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) as an 125 activating agent to promote amide bond formation between the support and the enzyme, and 126 iii) the addition of NaCl to desorb all the enzyme that was not covalently bound to the support. 127 After the immobilization, the derivatives were washed carefully. 128 The immobilization of TmCHMO was carried out by suspending the support in 25 mM MES 129 buffer (pH 6.0); then the enzyme was added to the suspension and left to adsorb ionically to 130 the support for 0.25 h. After that time, EDC was added to final concentrations of 25 or 35 mM 131 and left for 2 h. Finally, NaCl was added to a final concentration of 1 M and incubated for 1 h. 132 The immobilized derivative was washed with distilled water and filtered. 133 The immobilization of GDH-Tac was performed in 50 mM sodium phosphate buffer (pH 6.0). 134 The ionic adsorption step was completed after 0.5 h. A 200mM stock solution of EDC was 135 prepared, the pH was adjusted to 6.0 with HCl; different volumes were added to get final 136 concentrations of 1, 3, 5, 10 or 15 mM and incubated for 1h. Afterwards, NaCl was added to a 137 final concentration of 0.5 M and incubated for 0.5 h. Lastly, the support was washed gently 138 with 100 mM sodium phosphate buffer (pH 8.0) and filtered. 6 139 For the co-immobilization of TmCHMO and GDH-Tac, the support was suspended in 50 mM 140 sodium phosphate buffer (pH 6.0); both enzymes were added and incubated 0.25 h. After the 141 ionic step was completed, EDC was added to final concentrations of 10 or 20 mM and 142 incubated 1 h. NaCl was added to a final concentration of 1M. The derivative was washed with 143 distilled water and filtered. 144 The characterization of the immobilization was carried out by measuring the activity of the 145 supernatant and the suspension throughout the entire process, in order to determine the retained 146 activity (Equation (1)) and immobilization yield (Equation (2)). TmCHMO and GDH-Tac 147 immobilized on MANA-agarose were stored at 4 °C prior to use. Final suspension activity - Final supernatant activity 148 Retained activity (%)= ×100 (1) Initial supernatant activity Initial supernatant activity -Final supernatant activity 149 Immobilization yield (%)= ×100 (2) Initial supernatant activity 150 2.4.Determination of enzyme content 151 The cell lysate was pre-clarified by centrifugation (3220 g for 15 min.), and the total protein 152 content was determined by means of a Bradford Protein Assay Kit (Thermo Fisher Scientific, 153 Waltham, USA) using bovine serum albumin as standard. 154 Enzyme content was assessed using sodium dodecyl sulphate polyacrylamide gel 155 electrophoresis (SDS-PAGE) (NuPage 12%, Invitrogen, USA) ran in a Mini-PROTEAN II 156 apparatus (BioRad, USA) following the protocol of Laemmli et al.[36] Low range protein 157 markers were used for molecular weight determination. Gels were stained using Coomassie 158 G250 colloidal stain solution (34% v v-1 ethanol, 2% v v-1 H3PO4, 17% w v-1 NH4SO4 and 159 0.066% Coomassie G250) and Image LABTM software (BioRad, USA) was used for image 160 processing. 7 161 2.5. Determination of the reaction progress for biocatalyzed reactions using GC-FID 162 The substrate and product concentration were determined by GC-FID analysis in triplicate. 163 Aliquots of the reaction mixture (50 μL) were taken and diluted in acetonitrile (950 μL). The 164 sample was centrifuged using an Eppendorf centrifuge 5424 to remove precipitated protein and 165 analyzed by gas chromatography (GC-FID). The concentration of substrate and lactones were 166 determined using calibration curves. GC-FID analyses were performed using a Shimadzu GC- 167 2010 Plus Gas Chromatograph with a hydrogen flame-ionization detector and an SPB-1 168 capillary column (30 m × 0.25 μm × 0.25 mm inner diameter). For kinetics, the following 169 program was used: starting temperature of 60 ˚C maintained for 2 minutes, temperature 170 increased to 200 ˚C at a heating rate of 15 ˚C min-1 and then maintained at 200 ˚C for 2 minutes, 171 and temperature finally increased to 320 ˚C at a heating rate of 20 ˚C min-1 and maintained at 172 320 ˚C for 2 minutes (sample injected at 250 ˚C, with a split ratio of 10, 2 μL injection volume). 173 The following retention times were observed for kinetic samples measured from the reaction 174 mixture: 6.83 min for the substrate 1, 9.25 min and 9.36 min for the lactones 1b and 1a (Figure 175 1). 176 2.6.Reaction set-up and reaction conditions 177 The reactions were performed with a Metrohm 887 Titrino Plus titration apparatus. The pH 178 was monitored and adjusted to pH 8.0 by automatic addition of a solution of NaOH (1 M). The 179 reaction was performed in a double walled-glass and the temperature was maintained to 30 °C. 180 The reactions were performed in potassium phosphate buffer (25 mM), at pH 8.0. The reaction 181 was stirred at 500 rpm, and air was bubbled in the reaction volume at a rate of 8 mL min-1. 182 2.7.Bioreaction with soluble TmCHMO and GDH-Tac biocatalysts 8 183 The reaction vessel was loaded with 10 mM of 3,3,5-trimethylcyclohexanone (47.4 μL), 250 184 μM of NADP+ (5.9 mg), 350 mM of glucose, and 10% v v-1 of methanol (3 mL) for a total 185 reaction volume of 30 mL. The reaction was started by the addition of a 3.07% v v-1 of soluble 186 TmCHMO (0.921 mL of CFE containing 32.1 mg TmCHMO) and 4.87% v v-1 soluble GDH- 187 Tac (1.422 mL of CFE containing 65.8 mg soluble GDH-Tac). An additional 10 mM of 188 substrate (47.4 μL) was added every hour until a total of 140 mM of substrate. 189 2.8.Reusability of immobilized TmCHMO and GDH-Tac biocatalysts 190 The reaction vessel was loaded with 10 mM of 3,3,5-trimethylcyclohexanone (47.4 μL), 250 191 μM of NADP+ (5.9 mg), 30 mM of glucose, and 10% v v-1 of methanol (3 mL) for a total 192 reaction volume of 30 mL. The reaction was started by the addition of 5% v v-1 of immobilized 193 TmCHMO (20 mg TmCHMO g-1 support, 1.605 g of supported enzyme corresponding to 32.1 194 mg TmCHMO) and 5% v v-1 of immobilized GDH-Tac (29 mg GDH-Tac g-1 support, 1.605 g 195 supported enzyme corresponding to 46.5 mg GDH-Tac). The substrate and product 196 concentration were determined by GC-FID analysis in triplicate. At the end of the reaction, the 197 immobilized TmCHMO and immobilized GDH-Tac were filtered and washed with buffer. 198 New reaction medium containing 10 mM 3,3,5-trimethylcyclohexanone, 250 μM NADP+, 30 199 mM glucose and 10% v v-1 of methanol was prepared; to which the immobilized TmCHMO 200 and immobilized GDH-Tac rinsed with buffer were added to start the reaction. The supported 201 enzymes were stored at 4 °C overnight after cycles 5 and 10. 202 2.9.Reusability of the co-immobilized TmCHMO and GDH-Tac biocatalysts 203 The reactions were performed in a similar fashion as for the immobilized TmCHMO and GDH- 204 Tac biocatalyst. The biocatalyst concentration was 5.4% v v-1 (18.4 mg TmCHMO and 9.1 mg 205 GDH-Tac g-1 support, 1.74 g of supported co-immobilized enzymes corresponding to 32.1 mg 206 of TmCHMO and 15.83 mg of GDH-Tac). 9 207 3. RESULTS AND DISCUSSION 208 3.1.Biocatalyst immobilization on MANA-agarose support 209 Our goal was the oxidation of 3,3,5-trimethylcyclohexanone using the thermostable TmCHMO 210 (Figure 1). The NADPH cofactor was regenerated by applying GDH-Tac, which uses glucose 211 as a sacrificial cosubstrate. For this, both enzymes were evaluated in their soluble form as well 212 as immobilized on MANA-agarose (separately or co-immobilized). 213 Firstly, the immobilization of TmCHMO and GDH-Tac on MANA-agarose was studied aiming 214 to define the best conditions for the immobilization of the biocatalysts following two 215 approaches: separate enzyme immobilization and co-immobilization. Aiming to characterize 216 the immobilization processes these studies were performed at low activity loads to ensure no 217 mass transfer limitations once the enzyme is immobilized in the support (Table 1). 218 In order to obtain the highest immobilization yield and retained activity, the immobilization of 219 TmCHMO on MANA-agarose was assayed testing two different N-(3-dimethylaminopropyl)- 220 N’-ethylcarbodiimide (EDC) concentrations (25 and 35 mM). EDC is added once the protein 221 is ionically adsorbed to promote its covalent binding to the support. The results showed that 222 TmCHMO was completely adsorbed after 0.25 h and 35 mM of EDC was selected as the most 223 appropriate concentration allowing an immobilization yield of 93.0% and a retained activity of 224 62.4% (Table 1). 225 Regarding the immobilization of GDH-Tac, it was ionically adsorbed onto MANA-agarose 226 after 0.5 h. In the second phase of the immobilization, EDC was introduced at different 227 concentrations (1, 3, 5, 10, 15 and 20 mM) to promote the covalent binding of the enzyme to 228 the support. Among the EDC concentrations tested, 10 mM was chosen as the optimum as it 229 presented 78.7% immobilization yield and 57.1% retained activity (Table 1). 10 230 For the co-immobilization of TmCHMO and GDH-Tac, two EDC concentrations were tested, 231 10 and 20 mM. These values were selected taking into account the results obtained in the 232 previous immobilization studies. An EDC concentration of 10 mM was selected since GDH- 233 Tac retained activity was significantly affected by high EDC concentration. 234 3.2.Reutilization of the immobilized biocatalysts and comparison with the soluble 235 enzymes 236 The soluble and immobilized biocatalysts were applied to the oxidation of 3,3,5- 237 trimethylcyclohexanone. Similarly to our previous studies with TmCHMO and this 238 substrate,[15] it was necessary to control the pH during the reaction since each molecule of the 239 substrate that was converted resulted in the formation of one molecule of D-gluconolactone 240 which was hydrolyzed to gluconic acid and consequently increased the acidity of the reaction 241 (Figure 1). Auto-titration of the reaction by addition of NaOH at 1 M ensured a constant pH 242 throughout the reaction course. A co-solvent (10% v v-1 methanol) was added to aid the 243 solubility of the substrate, which is rather limited in water. This co-solvent was selected based 244 on our previous results showing that this co-solvent results in the fastest reaction rate compared 245 to other tested organic co-solvents.[15] 246 The oxidation of 3,3,5-trimethylcyclohexanone was first performed with both soluble 247 TmCHMO and GDH-Tac using a TmCHMO load of 1.07 mg mL-1 of reaction medium and an 248 enzyme ratio of 1:2.0 (mg TmCHMO mg GDH-Tac-1). Total conversion of the initial substrate 249 (10mM) was achieved in 1 h. Once the initial substrate was completely consumed, a fed-batch 250 strategy was applied by supplying an additional 10 mM of substrate to the reaction mixture 251 every hour up to a total of 150 mM of 3,3,5-trimethylcyclohexanone. The results showed that, 252 while the ketone was fully converted in 1 h for the first 3 substrate additions, the accumulation 253 of unreacted substrate was observed for the rest of the reaction until a final substrate 11 254 concentration of about 100 mM (Figure 2a). This change in the enzymatic reaction rate was 255 directly correlated to the amount of base needed to maintain the pH of the reaction, which is 256 related to the amount of gluconic acid co-product formed and substrate converted (Figure 2b). 257 The conversion for each addition was calculated and the obtained results are depicted in Figure 258 3. A sharp decrease in conversion per substrate addition was observed until an average 259 conversion of about 10% was observed. This was attributed to the loss of enzymatic activity 260 during the reaction, but substrate inhibition of TmCHMO as a consequence of substrate 261 accumulation in the reaction mixture probably also played a role. Product accumulation in the 262 reaction media could also contribute to a decrease in subsequent conversions since BVMOs 263 often exhibit product inhibition, as has been previously reported by other authors.[31,37,38] 264 Process metrics were analyzed for the fed-batch strategy using soluble enzymes (Table 2). The 265 total process time after 14 additions was 14.4 h with a final product amount of 0.308 g and a 266 final unreacted substrate amount of 0.423 g. The biocatalyst yields reached 9.6 and 4.7 mg of 267 product mg-1 of TmCHMO and GDH-Tac, respectively. 268 The performance of the TmCHMO and GDH-Tac which were separately immobilized at high 269 enzymatic loads was also studied. The TmCHMO immobilized derivative contained 20 mg of 270 monooxygenase g-1 of support, while the GDH-Tac derivative contained 29 mg of GDH-Tac 271 g-1 of support. Aiming to compare the results with the soluble enzymes, the reactions were 272 carried out using the same load of TmCHMO (1.07 mg TmCHMO per mL of reaction). The 273 ratio of TmCHMO/GDH-Tac was slightly lower (1:1.5) since it is determined by i) the 274 maximum immobilized derivative that can be used (10% v v-1) to ensure a proper suspension 275 and mixing and, ii) the enzymes load per mg of support obtained during the immobilization 276 processes. 12 277 Separately immobilized derivatives were used in the biooxidation reaction, where the first cycle 278 took about 1.33 hour to total substrate conversion (Figure 4a). The increase in reaction time for 279 a total conversion of the substrate during the first cycle could be related to i) the lower amount 280 of loaded GDH-Tac with the immobilized enzymes which could lead to the cofactor 281 regeneration reaction being the limiting step or/and ii) diffusion limitations of the NADP(H) 282 co-factor between the bead particles containing TmCHMO and GDH-Tac or /and iii) oxygen, 283 glucose or 3,3,5-trimethylcyclohexanone mass transfer limitations due to the diffusional 284 restriction of these molecules in the support particles. 285 The operational stability of the biocatalysts was studied. At the end of the reaction, both 286 immobilized enzymes were recovered and reused for conversion of additional substrate in the 287 same reaction conditions. In total, the immobilized enzymes were reused up to 15 times aiming 288 to compare the results with the data obtained using soluble enzymes where 14 additions were 289 carried out (Figure 4b). Full conversion was obtained for the first 5 cycles, after which the 290 conversion started to decrease slowly. 291 The process metrics obtained using separately immobilized biocatalysts are shown in table 2. 292 Even though the total reaction time of the process was 1.4-fold higher, the average final product 293 amount (0.422 g) increased by 37 %. Moreover, the use of separately immobilized enzymes 294 also improves the process performance by reducing in 2.1-fold the final unreacted substrate 295 amount (0.199 g) and increasing the TmCHMO biocatalyst yield by 36%. The overall 296 biocatalyst yield is increased by 74% due to the better performances obtained with the 297 separately immobilized biocatalysts, despite the lower GDH-Tac biocatalyst loading (70% of 298 the GDH-Tac loading of the reaction with the soluble enzymes). 299 The performance of the enzymes that were co-immobilized at high loads was also studied 300 (TmCHMO: 18.4 mg g-1 of support; GDH-Tac: 9.1 mg g-1 of support). In order to compare the 13 301 performance of the co-immobilized catalysts with the biocatalysts immobilized separately and 302 the soluble enzymes, the amount of co-immobilized support used in the oxidation reaction was 303 calculated so that the same amount of TmCHMO was applied in all cases (1.07 mg mL-1). The 304 ratio TmCHMO/GDH-Tac in this case (1:0.5) was determined by the ratio obtained during the 305 co-immobilization process, where both enzymes compete for the same support. 306 For this bioconversion, the reaction time was 1.17 h until the full conversion of the substrate, 307 17% higher compared to the soluble enzymes (Figure 5a). The higher reaction time compared 308 to the soluble enzymes could be due to the lower GDH-Tac load or to mass diffusional 309 restrictions, as already mentioned with the separately immobilized enzymes. However, even 310 though lower TmCHMO/GDH-Tac ratio was used when co-immobilized derivatives were used 311 (1:0.5) compared to the separately immobilized enzymes (1:1.5), the reaction time was 12% 312 lower. Thus, the reduction of the reaction time of the co-immobilized derivative compared to 313 the separately immobilized biocatalyst probably indicates that NADP(H) cofactor diffusional 314 restrictions between bead particles is likely the main cause of reaction time increase when 315 separately immobilized derivatives are used. 316 The operational stability studies were also carried out with the co-immobilized derivative 317 during 15 cycles (Figure 5b). Compared to the biocatalysts immobilized separately, the co- 318 immobilized biocatalysts performed much better with the re-uses. A substrate conversion of 319 58% was achieved for the last cycle (15) compared to 39% substrate conversion obtained for 320 the same cycle with the biocatalysts immobilized separately. 321 Regarding the process metrics (Table 2), co-immobilization, in particular, proved to be the best 322 option of this biotransformation with higher average conversion over all re-utilization cycles 323 (83%) despite the lower concentration of GDH-Tac in the reaction. The highest biocatalyst 324 yields and final average product amounts were achieved with the co-immobilized biocatalysts. 14 325 Comparing to the separately immobilized enzymes, all process metric analyzed were improved: 326 1.14-fold decrease in total process time, a 1.3-fold increase in final average product amount, a 327 1.4-fold decrease in the unreacted substrate, a 1.1-fold increase in average conversion, and a 328 1.3-fold increase in TmCHMO biocatalyst yield. The GDH-Tac biocatalyst yield was improved 329 by 3.7-fold because the experiment with the co-immobilized enzymes achieved the best 330 performances with the lowest GDH-Tac loading. 331 Compared to the soluble enzymes for which a fed-batch strategy was applied, even though the 332 total process time was slightly increased, the final average product formed was improved in 333 1.7-fold, the unreacted substrate amount decreased in 3-fold, the average final conversion was 334 increased in 1.6-fold, and the total biocatalyst yield was 3.6-fold higher. These values prove 335 the better performance of the co-immobilized enzymes in the target reaction studied compared 336 to separately immobilized enzymes. 337 4. CONCLUSIONS 338 TmCHMO was successfully immobilized on a MANA-agarose support with the co-enzyme 339 GDH-Tac to ensure co-factor regeneration. Both the enzymes immobilized separately and co- 340 immobilized displayed good retention of activity in repeated re-utilization for the oxidation of 341 3,3,5-trimethylcyclohexanone. Co-immobilized proved to give the most efficient biocatalyst 342 format, achieving the highest average conversion over 15 re-utilization cycles (83%) and a high 343 significant improvement of 3.6-fold of the total biocatalyst yield compared to the soluble 344 enzymes. Compared to the biocatalysts which were separately immobilized, a highest reaction 345 rate was observed which was attributed to more efficient diffusion of the NADP(H) co-factor 346 between the two enzymes immobilized on the same support. This work demonstrates that 347 immobilized BVMOs are promising biocatalysts for the synthesis of lactones, and in particular 348 polymeric building blocks. 15 349 ACKNOWLEDGMENTS. 350 The research for this work has received funding from the European Union (EU) project 351 ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and 352 Innovation actions H2020-LEIT BIO-2014-1. The views and opinions expressed in this article 353 are only those of the authors, and do not necessarily reflect those of the European Union 354 Research Agency. The European Union is not liable for any use that may be made of the 355 information contained herein. 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The enzymes were either immobilized on a MANA-agarose or 426 soluble. 427 428 429 430 431 432 433 434 18 435 Table 1. Overview of the characterization of the immobilization of TmCHMO and GDH-Tac 436 on MANA-agarose under optimum conditions. Enzyme Offered enzyme Immobilization Retained activity load* yield (%) (%) TmCHMO 5 U g-1 of support 93.0 62.4 (8 mg TmCHMO g-1 of support) GDH-Tac 5 U g-1 of support 78.7 57.1 (3.7 mg GDH-Tac g- 1 of support) Co-immobilized 5 U g-1 of support of 79.4 (TmCHMO) 12.9 (TmCHMO) TmCHMO and each enzyme GDH-Tac 96.5 (GDH-Tac) 48.2 (GDH-Tac) 437 *No substrate transfer limitations were found at this enzymatic load 438 439 440 441 442 443 19 444 445 446 Figure 2. a) Reaction course of the conversion of 3,3,5-trimethylcyclohexanone with soluble 447 TmCHMO and soluble GDH-Tac (TmCHMO/GDH-Tac 1:2.0) with the concentration of 448 substrate (blue circles) and product (black squares). The total amount of substrate accumulated 449 is shown with a pink dotted line. b) Profile of the volume of NaOH (1M) added during the 450 course of the reaction. The pink dotted line indicates the initiation rate of NaOH addition. 451 Reaction conditions: 10 mM of substrate initially + 10 mM every hour, 10% v v-1 methanol, 452 3.07% v v-1 soluble TmCHMO (1.07 mg mL-1), 4.87% v v-1 soluble GDH-Tac (2.19 mg mL- 1 453 ), 350 mM glucose, 250 µM NADP+. 454 20 455 456 457 Figure 3. Sequential additions of substrate for the reaction with soluble TmCHMO and soluble 458 GDH-Tac (TmCHMO/GDH-Tac 1:2.0) with conversion as a function of the number of 459 substrate additions (conversion = 1-([sub]f/[sub]i) with [sub]f the substrate concentration before 460 the next addition of substrate and [sub]i the substrate concentration after the last addition of 461 substrate). 462 463 464 465 466 467 468 469 21 470 471 Table 2. Overview of the performances of TmCHMO and GDH-Tac biocatalysts for the 472 oxidation of 3,3,5-trimethylcyclohexanone Biocatalyst Ratio Total Product Unreacted Average Biocatalyst yieldc format TmCHMO reaction formeda substratea convb (mg product/mg biocatalysts) :GDH-Tac time (g) (g) (%) (h) TmCHMO GDH-Tac Total Soluble 1:2.0 14.4 0.308 0.423 51 9.6 4.7 3.1 Immobilized 1:1.5 20.0 0.422 0.199 73 13.1 9.1 5.4 Co- 1:0.5 17.5 0.538 0.138 83 16.8 34.0 11.2 immobilized a 473 Cumulated amount of product and unreacted substrate (sum of each cycle for the 474 immobilized enzymes and value measured at the end of the reaction for the soluble enzymes) b 475 Average conversion calculated for 15 cycles for the immobilized enzymes and for 14 476 additions for the soluble enzymes. c Biocatalyst yield = total mg of product/mg of biocatalyst 477 (TmCHMO, GDH-Tac or TmCHMO + GDH-Tac). 478 479 480 481 482 22 483 484 Figure 4. Re-uses of TmCHMO and GDH-Tac immobilized on separate supports 485 (TmCHMO/GDH-Tac 1:1.5) with a) reaction profile for cycles 1, 6, 10, 15; and b) substrate 486 conversion after 1.33 hour for all cycles. The vertical dotted lines indicate overnight storage of 487 the immobilized enzymes in buffer solution. Reaction conditions: 10 mM of substrate, 10% v 488 v-1 methanol, 5% v v-1 immobilized TmCHMO, 5% v v-1 immobilized GDH-Tac, 30 mM 489 glucose, 250 µM NADP+, 1.33 h reaction time. 490 491 492 493 494 495 496 497 23 498 499 Figure 5. Re-uses of co-immobilized TmCHMO and GDH-Tac (TmCHMO/GDH-Tac 1:2.0) 500 with a) reaction profile for cycles 1, 6, 10, 15; and b) substrate conversion after 1.17 hour for 501 all cycles. The vertical dotted lines indicate overnight storage of the immobilized enzymes in 502 buffer solution. Reaction conditions: 10 mM of substrate, 10% v v-1 methanol, 5.4% v v-1 co- 503 immobilized TmCHMO and GDH-Tac, 30 mM glucose, 250 µM NADP+, 1.17 h reaction time. 24