This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 1 Co-immobilization of P450 BM3 and Glucose Dehydrogenase on different 2 supports for application as a self-sufficient oxidative biocatalyst 3 4 Short title: Co-immobilization of P450 BM3 and Glucose dehydrogenase on different supports 5 6 Jordi Solé1, Gloria Caminal2, Martin Schürmann3 and Gregorio Álvaro1, Marina Guillén1 a 7 Bioprocess Engineering and Applied Biocatalysis Group, Department of Chemical Biological 8 and Environmental Engineering, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. b 9 Institut de Química Avançada de Catalunya (IQAC) CSIC, Jordi Girona 18-26, 08034, Barcelona, 10 Spain c 11 InnoSyn B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands 12 13 Corresponding author: 14 Marina Guillén 15 Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de 16 Barcelona, 08193 Bellaterra, Catalonia, Spain 17 Telephone: +34 93 581 4793 18 Fax: +34 93 581 2013 19 marina.guillen@uab.cat 20 1 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 21 22 Abstract 23 BACKGROUND. The oxy-functionalization of non-activated carbon bonds by the bacterial 24 cytochrome P450 BM3 from Bacillus Megaterium, presents a promising field in biosynthesis 25 and it has gained much interest in recent decades. Nevertheless, the need for the expensive 26 cofactor NADPH, together with low operational stability of the enzyme have made the 27 implementation of this biocatalyst unfeasible in most cases for industry. 28 RESULTS. P450 BM3 and glucose dehydrogenase (GDH), as a cofactor regeneration enzyme, 29 were successfully co-immobilized obtaining a bi-functional self-sufficient oxidative biocatalyst. 30 Firstly, a broad screening on 13 different supports was carried out. Afterwards, five selected 31 agaroses with three different functionalities (epoxy, amine and aldehyde) were studied and 32 their immobilization processes optimized. Finally, P450 BM3 and GDH, were co-immobilized on 33 those supports showing the best performance for P450 BM3 immobilization: Epoxy-agarose 34 (Epoxy-agarose-UAB) presenting 83% and 20% retained activities respectively; AMINO-agarose 35 presenting 28% and 25% and Lentikats® with which both enzymes retained 100% of the initial 36 activity. Furthermore, the re-utilization of the self-sufficient immobilized derivatives was 37 tested in 5 repeated cycles. 38 CONCLUSIONS. P450 BM3 and GDH have been successfully immobilized on three supports and 39 their re-usability has been tested in a model reaction. It represents a step-forward for future 40 P450 BM3 industrial implementations. 41 42 2 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 43 44 45 Introduction 46 Cytochromes P450 (CYPs) are versatile monooxygenases able to hydroxylate non-activated 47 carbon bonds with the only requirement of molecular oxygen and an electron donor. CYPs are 48 found as: (i) double domain proteins, presenting a heme-containing oxidative part and a FAD- 49 /FMN-containing reductase; as (ii) triple domain enzymes with a third Ferredoxin subunit and 50 (iii) as fusion proteins with just one enzymatic unit [1]. These enzymes have been in the focus 51 of research in recent decades due to interest in their application as catalysts for the efficient 52 production of fine chemicals, polymers, active pharmaceutical ingredients or nutritional 53 supplements. They display a broad substrate range as well as the capability to catalyse a 54 variety of oxidations including epoxidations, hydroxylation of aromatics, N-oxidation, 55 deamination, dehalogenation and others [2]. 56 P450 BM3 (EC 1.14.14.1) from Bacillus megaterium was discovered in 1986 and excels among 57 CYPs because it presents the highest turnover numbers (17000 min-1 for arachidonic acid) [3], 58 [4]. In contrast with eukaryotic CYPs, P450 BM3 is a self-sufficient soluble protein that contains 59 both oxidative and reductase domains in the same polypeptide chain. Its natural substrates are 60 medium to long chain fatty acids, however, as with other CYPs, P450 BM3 is a promiscuous 61 enzyme. It accepts fatty amides and alcohols, hydroxylated fatty acids and ω-oxo fatty acids. It 62 also displays, not only hydroxylating activity, but olefin epoxidation, ring expansion, 63 heteroatom oxidation and dealkylation, and dehydrogenation across C-O, C-N and C-C bonds, 64 as well as carbon-carbon bond formation and cleavage [5]. 3 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 65 There are some limitations in the large-scale application of P450 BM3 such as, low operational 66 stability, a dependence on the expensive NADPH electron donor and low retained activity in 67 organic media [6]. Thus, there have been few cases where it has been attempt to be used 68 industrially [7], [8]. In order to tackle these bottlenecks, protein engineering, cofactor recycling 69 systems, surface modifications, reaction media engineering or immobilization are the main 70 strategies usually followed in biocatalytic processes. 71 Regarding the low operational stability, immobilization has proven to be an efficient 72 methodology for a broad type of oxidoreductases, not only for improving their stability but 73 also for enhancing process metrics by biocatalyst recycling [9], [10]. In addition, immobilization 74 can confer operational advantages such as the possibility to operate in continuous mode, 75 reduction of foam formation, increased stability in organic solvents or simplified product 76 purification [11], [12]. Immobilization of P450 BM3 has been pursued by other authors. For 77 example, Maurer et. al. tried to immobilize it on a variety of commercially available supports 78 [13]. They succeeded using the positively charged resins DEAE and SuperQ; and a Sol-Gel, but 79 only the last one was found suitable for bioconversions. Axarli et. al immobilized a triple 80 mutant onto Epoxy-sepharose, achieving retained activities of 81% and improved stabilities at 81 37°C [14]. Weber et. al. entrapped the heme-domain of the P450 BM3 into mesoporous 82 molecular sieves (MCM-41 and SBA-15) and found a correlation between the activity observed 83 from the derivative and pore diameter [15]. Furthermore, Zhao et. al. immobilized a mutant 84 on DEAE-650S, further entrapped into k-carrageenan together with catalase and zinc dust 85 (Zn/Co(III)sep) which served as electron source. They could operate the reactor during 10 86 batch cycles with continued conversions above 80% [16]. Other successful and more recent 87 examples involved the fusion of the enzyme to linkers that enabled immobilization on solid 88 surfaces [17]–[19]. From all the works present in the literature, few are the cases where P450 89 BM3 has been successfully immobilized covalently for bioconversion purposes. Many of the 4 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 90 above works focused on bio-sensing, in which low amounts of enzyme are required, or on the 91 electrochemical study of the enzyme [20], [21]. For large scale applications, the attachment of 92 large quantities of the enzyme to solid matrices by covalent bonds is a robust method that 93 often confers novel properties to the enzyme and it does not present leaching. 94 With regard to electron donors, there have been attempts to substitute the expensive NADPH 95 co-substrate for less costly products, e.g. NADH or H2O2 by protein engineering [22]–[24]. 96 Strategies for regenerating the expensive NADPH has been also studied both in enzymatic and 97 non-enzymatic manners [25]–[27]. The use of whole cell biocatalyst expressing P450 has also 98 been considered as a suitable and much cheaper strategy by taking advantage of the cellular 99 metabolism for NADPH regeneration [28], [29]. However, the use of whole cells entails several 100 drawbacks such as secondary undesired reactions or low reaction rates due to the substrate or 101 product mass transfer limitation. Among the different strategies for cofactor regeneration, the 102 utilization of secondary enzymes that make use of sacrificial substrates is one of the most 103 attractive systems. The use of enzymatic NADPH regeneration systems in P450-catalyzed 104 reactions has been reported by other authors. Different enzymes such as glucose 105 dehydrogenase, formate dehydrogenase or phosphite dehydrogenase proved to be efficient 106 and suitable biocatalysts for this purpose [13], [30], [31], [32]. 107 In this work, a bi-functional self-sufficient oxidative biocatalyst is obtained by the successful 108 co-immobilization of P450 BM3 and a GDH from Thermoplasma acidophilum as cofactor- 109 regenerating enzyme. Firstly, P450 immobilization was analysed prior to the selection of the 110 best co-immobilization strategy since P450 is a less robust enzyme compared to GDH. Thus, 111 deeper understanding of P450 BM3 immobilization by two different enzyme-support 112 interactions (adsorption or covalent attachment) is presented. In this sense, different supports 113 presenting a broad range of features were tested to get insight in the optimal immobilization 114 procedure that should be followed. Finally, co-immobilization of P450 BM3 and GDH was 5 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 115 performed by applying the best methodologies resulting for the P450 BM3. The encapsulation 116 in polyvinyl alcohol particles (Lentikats®) was also studied for the confinement of both P450 117 BM3 and GDH as an alternative to adsorption and covalent attachment strategies. 118 The resulting-self-sufficient biocatalysts were tested in terms of operational stability in the 119 hydroxylation of sodium laureate as a model reaction for proof-of-concept. 120 121 Experimental 122 Materials and supports 123 Nicotinamide adenine dinucleotide phosphate sodium salts in its oxidized and reduced form 124 (NADP+ disodium salt and NADPH tetrasodium salt) was purchased from BONTAC 125 Bioengineering (Shenzhen, Guandong, China). All the other reagents were purchased from 126 Sigma Aldrich® (St. Louis, MO, USA) and were of analytical grade if not stated elsewhere. 127 The supports used in the screening section were donated by Purolite® Life Sciences (Bala 128 Cynwyd, PA, USA) and their features are summarized in Table 2. 129 The Epoxy-agarose-SIGMA was purchased from Sigma-Aldrich®. According to the supplier’s 130 specifications it presented spherical shape, it had an activation degree of ≥ 20µmol/mL and 12 131 atoms spacer. 132 Non-functionalized agarose (spherical beads Ø 50-150µm) purchased from Agarose Bead 133 Technologies® (ABT®, Madrid, Spain) was used for obtaining Epoxy-agarose-UAB by support 134 functionalization carried out as described by Axarli et. al. [14]. The quantification of the epoxy 135 groups present on the supports was done following the method described by Gupta [33]. 6 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 136 Amino functionalized agarose (AMINO-agarose) presenting amino ethyl groups and aldehyde 137 functionalized agarose (GLYOXYL-agarose) with an extent of labelling of 40-60µmol/mL both 138 were also purchased from ABT®. All this information was always according to the supplier’s 139 specifications. 140 Lentikats® polymer was purchased from GeniaLab® (Braunschweig, Germany) and its 141 composition consists on a mixture of polyvinyl alcohol (10%), polyethylene glycol (6%) and 142 demineralized water (84%). 143 Recombinant expression of P450 BM3 and Glucose dehydrogenase in E. coli 144 P450 BM3 (CYP102A1 from Bacillus megaterium BM3) was recombinantly produced in 145 Escherichia coli in a 20 L scale batch fermentation employing an E. coli K12 derivative and a 146 pBAD/myc-HisC based expression vector. Glucose Dehydrogenase (GDH) from Thermoplasma 147 acidophilum (GDH-Tac) was co-expressed from the same vector in a poly-cistronic 148 arrangement of the GDH gene downstream of the P450 BM3 gene. A 500 ml pre-culture were 149 used to inoculate 20 kg main culture medium with 100 µg/ml neomycin. The pre-culture was 150 prepared in standard Luria-Bertani (LB) medium supplemented with 100 µg/ml neomycin. The 151 fermentation was performed using terrific broth (TB) medium with glycerol. 2.5 h after 152 inoculation of the fermenter as inducer, pre-sterilized L-arabinose was added to the fermenter 153 to final concentration 0.02 % (w/v). 24 hours after inoculation of the fermenter, the cell 154 material was harvested by centrifugation. Cell free extract was prepared by adding 2 weight 155 equivalents of 100 mM potassium phosphate (KPi) buffer (pH 7.0) to 1 weight equivalent of E. 156 coli wet cells and sonication of this cell suspension with an ultrasound probe for 20 min with 157 cooling on ice and centrifugation to remove the cell-debris. 158 Total protein and enzyme content 7 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 159 The cell lysate was pre-clarified by centrifugation (3220g for 15min.) and analysed accounting 160 for total protein content by means of Bradford Protein Assay Kit (Thermo Fisher Scientific, 161 Waltham, USA) using bovine serum albumin as standard [34]. 162 Enzyme content was assessed using the sodium dodecyl sulphate polyacrylamide gel 163 electrophoresis (SDS-PAGE) (NuPage 12%, Invitrogen, USA) run in a Mini-PROTEAN II apparatus 164 (BioRad, USA) following the protocol of Laemmli et. al. [35]. Low range protein markers were 165 used for molecular weight determination. Gels were stained using Comassie G250 colloidal 166 stain solution [34% (v/v) ethanol, 2% (v/v) H3PO4, 17% (w/v) NH4SO4 and 0.066% Comassie 167 G250] and the Image LABTM software (BioRad, USA) was used for image processing. 168 Active P450 BM3 content determination 169 P450 BM3 active form concentration was determined using the CO-differential spectra analysis 170 described by Omura and Sato, using ε = 91 mM-1 cm-1 [36], [37]. 171 P450 BM3 activity measurement 172 The activity of the P450 BM3 was measured spectrophotometrically (λ=340nm) following the 173 consumption of NADPH (ε= 6.22 mM-1 cm-1, 200µM), using sodium dodecanoate (sodium 174 laurate) as substrate (1,3 mM dissolved in sodium phosphate buffer 50mM pH=7,5) and the 175 correspondent amount of sample [1/20 of the total volume (VT)]. The basal consumption of 176 NADPH by unspecific enzymes present in the lysate was determined by this same test but 177 avoiding the addition of the substrate and adding buffer instead. This consumption rate is 178 subtracted from the measurement with sodium dodecanoate. One unit of activity (UA) is 179 defined as the enzyme required to convert 1µmol of NADPH per minute at those given 180 conditions (30°C and pH = 7.5). The absorbance was recorded using a spectrophotometer Cary 181 50 Bio UV-visible (Palo Alto, USA). The equipment allows temperature control and stirring. 182 Plastic micro cuvettes BRAND® UV (Sigma Aldrich®) were used for soluble enzyme 8 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 183 characterization and 3.5mL quartz cuvettes HELLMA® 100-QS (Hellma Analytics, Müllheim, 184 Germany) with magnetic stirring, were used for the immobilized derivatives (suspension). In 185 the first case a total volume of 500µL was reached while in the second it was 2mL. 186 Glucose dehydrogenase activity measurement 187 GDH activity is measured spectrophotometrically (λ=340nm) following the formation of 188 NADPH. Certain amount of sample (1/20 of the VT) was added to a solution containing D- 189 Glucose (200mM dissolved in sodium phosphate buffer 100mM pH=8) and NADP+ (400µM). 190 The same procedure explained above was applied for measuring the non-GDH formation of 191 NADPH and the same consideration is taken to obtain the UAs. The absorbance was also 192 recorded using the aforementioned equipment and conditions. 193 Immobilization metrics 194 In every immobilization, a characterization was pursued in order to obtain the retained activity 195 (equation 1) and immobilization yield (equation 2). Supernatant and suspension activities 196 were analysed over time until a steady state was reached and in all cases the activity of a blank 197 (no support) was also monitored to ensure that the enzyme activity was not affected by 198 protocol’s conditions. A 10% w/v relation between the support and the total volume was 199 always stablished and small amounts of enzyme were loaded (4-5UA of P450 BM3/g of support 200 and 5-6UA of GDH/g of support) so that diffusional limitations were minimized. 𝑈𝐴 𝑈𝐴 𝑆𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( )−𝑆𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( ) 𝑔 𝑔 201 𝑅𝑒𝑡𝑎𝑖𝑛𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = 𝑈𝐴 𝑥 100 (Equation 1) 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑜𝑓𝑓𝑒𝑟𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( ) 𝑔 𝑈𝐴 𝑆𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( ) 𝑔 202 𝐼𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 (%) = (1 − 𝑈𝐴 ) 𝑥 100 (Equation 2) 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑜𝑓𝑓𝑒𝑟𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( ) 𝑔 9 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 203 For the assessment of the loading capacity the supports were saturated with enzyme and the 204 activity was calculated taking into account that diffusional limitations do not allow the actual 205 visualization of the activity in the support (equation 3). 𝑈𝐴 𝑈𝐴 206 𝐿𝑜𝑎𝑑𝑖𝑛𝑔 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 ( 𝑔 ) = (𝑂𝑓𝑓𝑒𝑟𝑒𝑑 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( 𝑔 ) − 𝑈𝐴 𝑅𝑒𝑡𝑎𝑖𝑛𝑒𝑑 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) 207 𝑆𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( )) 𝑥 (Equation 3) 𝑔 𝐼𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑎𝑡𝑖𝑜𝑛 𝑦𝑖𝑒𝑙𝑑 (%) 208 Moreover, the amount of protein per gram of support was also calculated, analysing the initial 209 protein content and subtracting from it, the concentration still remaining in the supernatant at 210 the end of the process. In all cases protein concentration was determined by means of 211 Bradford Protein Assay Kit, as already described. 212 Immobilization of P450 BM3 onto Purolite® resins 213 From Purolite®, a set of commercially available resins presenting a variety of matrices, pore 214 sizes, linker lengths, and functional groups (Table 2) were tested for the immobilization of 215 P450 BM3. The conditions in which immobilizations were carried out in each case were those 216 specified by the supplier’s protocols. In all cases, the relation of 10% w/v between support and 217 total volume was maintained. The immobilizations were done at 25°C and mild agitation. The 218 incubation times ranged from 0.5 to 5 hours depending on the speed at which the enzymes 219 were attached. The epoxy containing methacrylates (ECR8204F, ECR8215F and ECR8285) were 220 used in 50mM sodium phosphate buffer pH 8 containing 0.5M NaCl. The amino functionalized 221 resins (ECR8309F, ECR8315F, ECR8409F and ECR8415F) were mixed with the enzyme in 50mM 222 sodium phosphate buffer pH 6. The supports made of a mixture of styrene and methacrylate 223 (ECR1061M and ECR1030M) were utilized in 50mM sodium phosphate buffer pH 7.5; finally, 224 immobilizations using the epoxy agaroses (Praesto 45, 65 and 90) were performed in 1M 225 potassium phosphate buffer pH 8. 10 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 226 Immobilization of P450 BM3 onto Epoxy-agarose-SIGMA and Epoxy-agarose-UAB supports 227 For the other epoxy-agaroses (Epoxy-agarose-SIGMA and Epoxy-agarose-UAB), the enzyme 228 was dissolved in 1M (pH 8) potassium phosphate buffer, mixed with the resin (10% w/v) and 229 left at 25°C with mild agitation. The Epoxy-agarose-UAB was incubated for 4 hours and Epoxy- 230 agarose-SIGMA was incubated for 0.5h. At the end of the process, in order to eliminate the 231 unreacted epoxy groups, the samples were incubated with 0.2M β-mercaptoethanol for at 232 least 2h at 25°C. 233 Immobilization of P450 BM3 onto GLYOXYL-agarose support 234 For the immobilization using GLYOXYL-agarose different pH’s were tested (6.5 – 8.5) using 235 50mM sodium phosphate buffers. The immobilizations lasted 6 hours and were done at 25°C 236 and mild agitation. For the reduction of the Schiff base, two different reducing agents were 237 studied: sodium borohydride (1mg/mL) added at the end of the immobilization and sodium 238 cyanoborohydride (0.05mg/mL for characterization and 0.4mg/mL for high loads of enzyme) 239 added at the beginning of the immobilization. 240 Immobilization of P450 BM3 onto AMINO-agarose support 241 For AMINO-agarose 50mM sodium phosphate buffer (pH 6) was used to dissolve the enzyme 242 and mix it with the support. Coming up next, the enzyme was left to ionically adsorb onto the 243 support for 0.5h-1h. After that, N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) was 244 added and incubated for 30min. at different concentrations to activate the carboxylic groups 245 and promote the covalent binding. The concentration of EDC was optimized in the 246 characterization phase (1, 2, 3, 4, 5, 10, 15, 20mM) and also when using high loads of enzyme 247 (3, 6, 12, 30, 60mM). Finally, 0.5M NaCl was introduced to desorb all the protein attached non- 248 covalently. The immobilization was carried out at 25°C and mild agitation. 11 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 249 Co-immobilization of P450 BM3 and GDH in AMINO-agarose and Epoxy-agarose-UAB 250 The conditions in which immobilizations were carried out were exactly the same as previously 251 described for P450 BM3 for AMINO-agarose and Epoxy-agarose-UAB. The activity of the GDH 252 present in the lysate was also monitored in this stage together with the P450 BM3. Retained 253 activities and immobilization yields were obtained for GDH and P450 BM3. 254 Co-Immobilization of P450 and GDH into polyvinyl alcohol lenses (Lentikats®) 255 Co-immobilization of GDH and P450 BM3 by entrapment was carried out using an organic 256 polymer commercialized as Lentikats®. The solid mixture was heated up to 95°C until it turned 257 into a transparent liquid. Then it was cooled down to 40°C and 8mL of the polymer were mixed 258 with 2mL of a suspension containing the desired amount of biocatalyst. Afterwards the 259 mixture was poured on a petri dish and the lenses were printed using the LentiPrinter®. The 260 resulting plates were left to dry at 25°C for 1h until they lost 80% of its weight approximately. 261 Once printed, the support adopted a lentil-like shape of 1-2mm diameter and 200-300µm wide 262 where the proteins were entrapped inside. 263 Re-usability of the biocatalyst for the hydroxylation of sodium laureate 264 The utilization of the immobilized enzymes in repeated reaction cycles were studied following 265 the consumption of sodium laurate. The reactor (VT=5mL) contained 1.53mM sodium laurate, 266 0.2mM NADP+ and 12.5mM D-glucose. All dissolved in 50mM sodium phosphate buffer pH 7.5. 267 The enzyme added was the minimum necessary to convert at least 90% of the initial substrate 268 in 1hour. The reactors used were purchased from MultiSynTech (Witten, Germany) and 269 consisted of 10mL plastic vessels with a porous plate at the bottom. It allowed the extraction 270 of the whole reactor liquid content by filtering with this same porous plate without losing any 271 fraction of the immobilized derivative. The reaction took place at 25°C and 1000 rpm using a 272 Multi-Therm H5000-HC-E thermo-shaker (Benchmark Scientific Inc., Sayreville, NJ, USA). After 12 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 273 each cycle, the immobilized enzymes were washed twice with sodium phosphate buffer 50mM 274 pH 7.5 then new reaction medium was added and substrate conversion was quantified. 275 Experiments were carried out in duplicate. 276 GC-FID analysis of sodium laurate 277 Samples containing sodium laurate and its products were analysed using a 7890A gas 278 chromatograph (Agilent Technologies, USA) equipped with a HP-INNOWAX 19095N-123 279 column (30m, 0.53mm, 1µm, Agilent Technologies). Reaction samples were filtered (Ø 0.22µm) 280 and 50µL of hexanoic acid (2.8mg/mL), that served as internal standard, were added to 300µL 281 of sample. The column temperature started at 150 °C, increased to 240 °C at 24 °C/min. and it 282 was held at final temperature for 11 minutes. The injector temperature was kept at 300 °C; for 283 the flame ionization detector, the temperature was 320 °C. Helium was used as a carrier gas at 284 a flow rate of 8 mL/min. All analyses were carried out in duplicate. 285 286 Results and discussion 287 Characterization of the E. coli lysate containing P450 BM3 and GDH 288 As already mentioned, P450s are enzymes that require a cofactor to perform the redox 289 reactions they catalyse. One of the strategies widely used for overcoming the disadvantages of 290 co-factor dependency is the use of an enzyme-coupled reaction for the regeneration of the 291 expensive NADPH. In this work, GDH (EC 1.1.1.47) from the archaea Thermoplasma 292 acidophilum was used as NADPH-recycling enzyme using glucose as sacrificial substrate. This 293 robust and stable tetrameric protein is reported to be an efficient enzyme for NADPH 294 regeneration [31], [32], [38]. 13 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 295 In the present work, GDH was co-expressed with P450 BM3 in E. coli and both enzymes were 296 present in the cell lysates utilized. 297 The characterization of the lysate was performed regarding protein content, enzyme content 298 (SDS-PAGE), activity and P450 BM3 active form concentration and the results are shown in 299 Table 1. 300 The P450 BM3 was expressed at a higher level than the GDH, however, under the conditions in 301 which the activity tests were performed, GDH gave higher specific activity. Table 1, also shows 302 the correlation between Units activity (UAs) and concentration (nmols) of active enzyme; for 303 the P450 BM3 case, 2.85 UA/nmol. 304 Screening of supports for the immobilization of P450 BM3 – Purolite® 305 The main objective of the present work was to obtain a bi-functional self-sufficient oxidative 306 biocatalyst by co-immobilization of P450 BM3 and GDH. To accomplish this, supports and 307 immobilization conditions that were found to be most suitable for the P450 BM3 enzyme were 308 prioritised, since cytochrome is the key enzyme performing the target reaction and it has been 309 described as an unfavourable enzyme for immobilizing. GDH, on the contrary, has been 310 described as a robust and more stable enzyme so difficulties in its immobilisation were not 311 anticipated [38]. In fact, successful immobilization of GDH on different kinds of support has 312 been reported by several authors [39]. The results obtained for P450 BM3 immobilization, the 313 materials tested and its features are summarized in Table 2. The supports purchased in the 314 screening kits encompass a range of different particle sizes (45µm to 710µm), matrices 315 (methacrylate, styrene and agarose), functional groups (amino, epoxy and aldehyde), enzyme- 316 support interaction (covalent binding or hydrophobic adsorption) and linker lengths (2 to 18 317 carbon atoms). All of these properties have been identified as critical for successful enzyme 318 immobilization [12]. 14 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 319 As shown in Table 2, the immobilization yields obtained were higher than 90% in all cases, 320 except for the supports that interact with the enzyme by hydrophobic adsorption: ECR1061M 321 and ECR1030M. In both cases the immobilization yields only reached 21% and 33% 322 respectively. On the other hand, no retained activities or very low values (<20%) were 323 observed in all tested supports, except for Praesto 45, 65 and 90 (Entries X to X; all epoxy- 324 agarose supports). According to the titration performed, Praesto 45, 65 and 90 all contained an 325 epoxide concentration of 9, 6 and 8µmol per gram of support respectively and resulted in 326 retained activities of 60-68% after 4h incubation. The maximum loading capacities of these 327 epoxy-agarose resins ranged from 18 to 20UA/g and 7 to 8mg protein/g of support. The 328 immobilization profiles were very similar among the three supports and they did not show any 329 significant correlation with the particle size or the initial epoxide concentration (Fig. 1A). 330 Regarding the methacrylate and styrene matrices, even though different ionic strengths and 331 pH values were assessed, none of the tested supports showed retained activities above 20%. 332 P450 BM3 had adhered to the matrices in most cases, but did not remain active. Two 333 methacrylate-based materials harbouring amino groups on six carbon chain linkers (ECR8409F 334 and the ECR8415F) gave retained activities of 24% and 22% respectively when the enzyme was 335 adsorbed onto the resins solely by ionic interactions, however once carbodiimide was added, 336 to promote covalent bond formation, the activity was reduced to 6% and 18% respectively. 337 Furthermore, following the supplier’s protocol, the four amino methacrylate based supports 338 were further functionalized with glutaraldehyde prior to enzyme binding, but none resulted in 339 successful immobilization either. 340 It should be also mentioned that methacrylic matrices with the same functional groups than 341 the epoxy-agarose support (ECR8204F, ECR8215F and ECR8285) did not lead to actively 342 immobilized enzyme, as can be seen by the low retained activities obtained (0%, 2% and 3% 343 respectively). These results could indicate that for P450 immobilization the hydrophilicity of 15 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 344 the matrix is a key factor. The higher hydrophobicity of the methacrylate/styrene matrices, 345 together with the flexible nature of the enzyme and the presence of two active sites, are 346 probably the causes behind the deactivation of the biocatalyst [40]. This hypothesis would be 347 also in accordance with the low immobilization yields and retained activities obtained with the 348 styrene matrices (ECR1061M and ECR1030M) where, because the immobilization mechanism 349 is also based on hydrophobic adsorption to the highly hydrophobic nature of the matrix, both 350 the immobilisation yield and retained activity are low. Other authors have also reported 351 unsuccessful attempts to immobilize P450s by hydrophobic adsorption [13]. 352 Taking into account the obtained results in the screening which showed a strong effect of the 353 hydrophilicity of the matrix on P450 immobilization, agarose was chosen to perform further 354 studies. 355 Immobilization of P450 BM3 onto different functionalized agaroses 356 In addition to the epoxy resins already tested (Praesto 45, 65 and 90), two other agarose 357 matrices with free epoxy groups and different expoxide concentrations were studied: a 358 commercial lyophilized epoxy-agarose resin from Sigma-Aldrich® (Epoxy-agarose-SIGMA) and 359 an in-house functionalized agarose resin (Epoxy-agarose-UAB) prepared as shown in the 360 experimental section. In addition an agarose based matrices containing aldehyde (GLYOXYL- 361 agarose) and primary amine (AMINO-agarose) functional groups were tested. 362 The Epoxy-agarose-SIGMA resin, according to the titration performed, contained an epoxide 363 concentration of 110µmol/g and gave an immobilization yield of greater than 95% after 0.5h 364 (Figure 1B). However, a rapid decrease in activity of the suspended biocatalyst was noted 365 during the immobilization procedure, indicating a strong deactivation of the enzyme after 366 immobilization. Thus, after just half an hour, low retained activities (25%) were observed from 367 this support. These results could indicate that the utilization of an epoxy-agarose with high 16 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 368 epoxide concentration, such as the Epoxy-agarose-SIGMA, results in a progressive loss of 369 activity over time due to the multipoint attachment of the enzyme to the support [41]. The 370 explanation can be found in the reactivity of the epoxy groups which differs over time: firstly, 371 reacting with the amines present on the surface of the enzyme and secondly reacting with the 372 carboxyl and thiol groups and [42]. At the end, if the incubation time is long enough, the 373 protein can be attached by too many points and lose its whole catalytic activity. This issue can 374 be stopped adding a blocking agent that removes the unreacted epoxy groups once the 375 protein is immobilized. In Figure 1B, β-mercaptoethanol (0.2M final concentration) was added 376 after 0.5h and the suspension was incubated for 3.5h at 25°C. No significant loss of activity was 377 observed during this period which validates the hypothesis of the multipoint attachment as 378 the main reason for enzyme deactivation. However, due to its elevated cost (118€/g) and low 379 retained activities, compared to the Praesto supports, the Epoxy-agarose-SIGMA was 380 discarded from further studies. 381 On the other hand, the Epoxy-agarose-UAB displayed an epoxide concentration of 30µmol/g 382 and gave more than a 95% immobilization yield and 83% retained activity after 0.5h (Figure 383 1C), with a maximum loading capacity of 30UA/g (7.5mg protein/g support). A decrease in 384 retained activity can be also seen, reaching 50% after 4h of incubation probably due to the 385 excessive number of covalent bindings. In this case, as well as for Praesto supports (Figure 1A), 386 the agarose contains a lower epoxide concentration compared to Epoxy-agarose-SIGMA. 387 Therefore, the activity loss due to the multipoint attachment is lower for the Epoxy-agarose- 388 UAB or even negligible for the Praesto compared to the Epoxy-agarose-SIGMA. Thus, when 389 choosing the right support, a consensus should be found. Higher epoxide concentration may 390 imply higher loading capacity as is seen when comparing Praesto (18 to 20 UAs/g) and Epoxy- 391 agarose-UAB (30UAs/g); however, at the same time, higher epoxide concentration entails 392 higher deactivation of the enzyme over time as it is the case comparing all three epoxy- 17 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 393 agaroses: Praesto (32-40% activity lost), Epoxy-agarose-SIGMA (75% activity lost) and Epoxy- 394 agarose-UAB (50% activity lost). In this sense, due to the loading capacity, the high retained 395 activities reached and the low cost (2.5€/g), the Epoxy-agarose-UAB was considered the best 396 candidate for the immobilization of P450 BM3 among epoxy-agaroses. This study represents a 397 deeper insight into the work of Axarli et. al. [14] in the understanding of the immobilization of 398 P450 BM3 on epoxy-agaroses and sets the bases for an optimal process. 399 As mentioned before, other agarose-based supports with different functional groups were also 400 tested such as GLYOXYL-agarose and AMINO-agarose. The GLYOXYL-agarose (aldehyde 401 activated) immobilization must be pursued in alkali media [43], however, P450 BM3 is not 402 stable above pH 8.5 (data not shown). After screening a range of immobilization pH’s (6.5 – 403 8.5), 7.5 was the best performing one resulting in 50% retained activity after 6h incubation 404 (data not shown). For the reduction of the Schiff base formed between the aldehyde of the 405 resin and the amine of the enzyme, two reducing agents were tested: i) sodium borohydride 406 (1mg/mL) which is added at the end of the incubation and ii) sodium cyanoborohydride 407 (0.05mg/mL) which has slower kinetics and can be added at the beginning of the 408 immobilization. Sodium borohydride resulted in a complete deactivation of the enzyme whilst 409 the sodium cyanoborohydride strategy resulted in 36% of retained activity. Thus, sodium 410 cyanoborohydride was selected as the most suitable reducing agent. Regarding the studies on 411 maximum loading capacity, the values previously obtained in the characterization (36% 412 retained activity) at low enzyme loadings were not extrapolated and a major inactivation 413 occurred. This support has proved suitable for the immobilization of P450 BM3, nevertheless, 414 since the covalent immobilization using high loading was not successful, it was discarded from 415 further studies. 416 Regarding AMINO-agarose, as for the other tested supports functionalized by amino groups 417 (i.e. ECR8309F, ECR8315F, ECR8409F and ECR8415F) the immobilization is based on a three- 18 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 418 stage process. In the first stage, the enzyme is adsorbed onto the support surface by ionic 419 interaction of the amino groups of the support and the carboxyl groups of the enzyme, thus an 420 ionic adsorption occurs. In the second stage, carbodiimide is added to the medium aiming to 421 activate the carboxyl groups present on the enzyme making covalent bonding possible. 422 Solutions with high ionic strength such as sodium chloride (0.5M) can be finally added to 423 desorb all the protein attached non-covalently (third stage). Ionic adsorption on AMINO- 424 agarose was performed at pH 6 and gave a 58% retained activity (Figure 1D phase I). This value 425 was 2.8-fold higher than the retained activities obtained during the ionic adsorption for the 426 methacrylate based materials ECR8409F and ECR8415F harbouring the same functional group 427 (21%), as previously discussed. The maximum loading capacity achieved was 112UA/g (50mg 428 protein/mL) which represents the highest value among all the resins tested. Covalent binding 429 of the amine and the carboxyl groups was carried out by adding EDC following ionic adsorption 430 phase (Figure 1D phase II). Since carbodiimide can lead to enzyme deactivation, different 431 concentrations and incubation times were studied to determine the optimal conditions for the 432 covalent binding phase. These studies showed optimal values of 3mM EDC and 30min reaction 433 time respectively. Following covalent attachment, the biocatalyst was incubated with 0.5M 434 NaCl for 2h (Figure 1D, phase III) in order to increase the ionic strength of the media and 435 release all the protein not adhered covalently. 28% of the initial activity offered was covalently 436 bound to the agarose, which represents 30% less activity than the value obtained for the ionic 437 adsorption phase (Figure 1D, phase I). On the other hand, compared to the methacrylate- 438 based supports, the retained activity was 4.7-fold and 1.6-fold higher than the obtained with 439 ECR8409F and ECR8415F respectively. Regarding the maximum load, 53UA/g was reached (47 440 mg protein/mL), representing a 52% decrease in immobilized activity compare to the obtained 441 by ionic adsorption, likely due to enzyme deactivation caused by the carbodiimide. When 442 testing for the maximum loading capacity the concentration of EDC was further optimized 19 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 443 since more enzyme was added (Figure 2). It resulted in an optimal concentration between 6 444 and 12mM. This second value was chosen due to the higher immobilization yield that it 445 entailed. This support represents a step forward in comparison with the other tested agaroses 446 and it is the first work reporting a successful covalent immobilization of P450 BM3 in an 447 amino-based matrix. 448 Co-immobilization of P450 BM3 and Glucose dehydrogenase 449 As it has been already mentioned, for co-immobilization of both GDH and P450 BM3, it was 450 considered to prioritize the supports and immobilization conditions that were found to be 451 suitable for P450 BM3, i.e. Epoxy-agarose-UAB and AMINO-agarose. 452 The immobilization of GDH using Epoxy-agarose-UAB resulted in poor affinity of the protein for 453 the support with immobilization yields below 20%. However, of the enzyme that did adhere, a 454 slight over-activation was observed compared to the same quantity of free enzyme. At the end 455 of the incubation (4h), retained activity of 20% and a maximum loading capacity of 3UA/g were 456 achieved. Since the conditions were exactly the same as reported above for P450 BM3, the 457 activity for the cytochrome was comparable and resulted in 50% retained activity and 30UA/g 458 (7.5mg protein/g support). 459 With AMINO-agarose, GDH presented high affinity for the support and it also showed an over- 460 activation effect when adhered to the matrix. It had better tolerance to the presence of EDC 461 compared with P450 BM3, with 10mM being the optimal concentration for this enzyme (57% 462 retained activity) in the characterization phase. Coimmobilisation of both enzymes, initially 463 using a low carbodiimide concentration of 3mM, resulted in a 25% retained activity for GDH 464 and 28% for P450 BM3. Using a higher loading of EDC (12mM) resulted in a loading capacity of 465 18 UA/g for GDH and 53 UA/g for P450 BM3. In summary, a self-sufficient oxidative biocatalyst 466 was obtained. 20 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 467 Co-immobilization of P450 BM3 and GDH into polyvinyl alcohol particles - Entrapment 468 Finally, as an alternative to immobilization by adsorption or by covalent attachment, an 469 entrapment strategy was also investigated for co-immobilization. Both P450 BM3 and GDH 470 were successfully embedded into Lentikats® (polyvinyl matrix) as described in the 471 experimental section. The analysis undertaken, showed maximum retained activities of 100% 472 for both P450 BM3 and GDH. 473 In the sense of the loading capacity employed, the activity and concentration of protein in the 474 polymer was 1.8UA/g of P450 BM3, 0.8UA/g of GDH and 1.1mg protein/g respectively. It was 475 not further increased due to operational issues. The capacity is limited by an aggregation effect 476 observed when charging high concentrations of lysate with which the lenses form a compact 477 mass that cannot be used in our reactions. This entails a serious drawback when looking to 478 industrial feasibility because the enzyme should be previously purified or the specific activity 479 of the support would remain too low [28]. However, since the immobilization had been 480 successful, albeit with low enzyme loading, and it is the first work that reports a successful 481 entrapment of both P450 and GDH in Lentikats ®, it was decided to continue working with this 482 immobilized derivative. 483 Reusability of the biocatalyst using sodium laurate as substrate 484 From all the supports tested, AMINO-agarose, Epoxy-agarose-UAB and Lentikats® were 485 considered good candidates to be potential carriers in bio-catalysis applications. It is well- 486 known that uncoupling effects on P450s produce reactive oxygen species that inactivate the 487 enzyme (uncoupling) [44]. This handicap is less relevant when working with native substrates 488 such as sodium laureate and so this substrate was used as a model for studying the reusability 489 of the self-sufficient biocatalyst. Firstly, the coupling efficiency of P450 towards this substrate 21 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 490 was determined and then reusability studies were performed using the self-sufficient 491 P450/GDH biocatalysts obtained with the aforementioned carriers. 492 Reactions using sodium laureate (1.3mM) and NADPH (0.2mM) were performed, monitoring 493 and comparing substrates consumption by means of GC-FID. A coupling efficiency of 90.4% 494 was observed, which means that, 90.4% of the NADPH consumed was employed for sodium 495 laurate hydroxylation, with the remainder directed towards uncoupled reactions. This 496 substrate was considered adequate to assess the operational stability of the enzymes in 497 repeated reaction cycles. 498 Total turnover number (mols of substrate consumed/mols of P450 BM3) (TTN) was chosen as 499 the metric to evaluate the yield of the self-sufficient biocatalyst in comparison with the soluble 500 lysate. To assess it, the immobilized P450 BM3/GDH (Epoxy-agarose-UAB, AMINO-agarose and 501 Lentikats®) biocatalysts were tested in sodium laurate reaction cycles of 1 hour each utilizing 502 glucose as the sacrificial substrate and the cofactor in its oxidized form (NADP+) thus forcing 503 the cofactor regeneration to start before the P450-catalyzed reaction. Moreover, NADP+ 504 starting concentration was 6.5-fold lower than the sodium laureate., The amount of P450 BM3 505 required to convert > 90% of sodium laurate in 1 hour was found to be 0.18-0.2UA/mL (0.063- 506 0.07 nmols/mL). GDH on the other hand, was always ensured to be present in excess so that it 507 did not limit the hydroxylation reaction. The conversions obtained in each cycle for the 508 immobilized systems are presented in Figure 3. The TTN for the soluble reaction was 21,500 509 mols of sodium laurate consumed/mol of P450 BM3 in 1 hour of reaction. The immobilized 510 derivatives allowed the re-utilisation of the self-sufficient biocatalyst boosting the TTN by 2.31- 511 fold for the Epoxy-agarose-UAB, 2.98-fold for AMINO-agarose and 2.3-fold for the enzymes 512 immobilized into Lentikats®. As shown in Figure 3, the conversion dropped in an exponential 513 manner for epoxy-agarose (Figure 3A). On the contrary, with AMINO-agarose and Lentikats® 22 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 514 the decay was more linear (Figure 3B). At the end, all three derivates could be re-used 515 successfully, thus demonstrating a significant operational stability. 516 517 Conclusions 518 P450 BM3 has gained much interest in recent years in biocatalysis for its oxy-functionalization 519 characteristics, although, the implementation of this enzyme in industrial processes has lacked 520 optimal metrics. Operational stability as well as its requirement of NADPH in stoichiometric 521 amounts have been identified as the main bottlenecks. Aiming to overcome these two main 522 drawbacks, P450 BM3 and GDH have been co-immobilized in the present work to obtain a 523 robust bi-functional self-sufficient biocatalyst. 524 P450 BM3 and GDH were covalently co-immobilized on epoxy-functionalized agarose and 525 amino-functionalized agarose. Both enzymes were also successfully co-immobilized by 526 entrapment in a polyvinyl alcohol/polyethylen-glycol matrix named Lentikats®. From the five 527 epoxy-agaroses tested, the optimal support (Epoxy-agarose-UAB) presented an epoxide 528 concentration of 30µmol/g and retained activities of 83% for P450 BM3 and 20% for GDH. On 529 the other hand, AMINO-agarose showed retained activities of 28% for P450 and 25% for GDH. 530 Finally, the entrapment of both enzymes in Lentikats ® lead to P450 BM3 and GDH co- 531 immobilization maintaining 100% of the activity initially loaded. 532 P450 BM3 was also immobilized on aldehyde-functionalized agarose and presented 45% 533 retained activity. However, this support did not allow high enzyme loads required for reaction 534 testing, thus making this support unattractive for further studies. 535 The operational stability of the obtained bi-functional self-sufficient biocatalysts (Epoxy- 536 agarose-UAB, AMINO-agarose and Lentikats®) was analysed in terms of re-usability for sodium 23 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 537 laureate oxidation. All three derivatives showed successful reuse in 5 cycles, entailing an 538 increment in the TTN of 2.31-fold the Epoxy-agarose-UAB, 2.98-fold the AMINO-agarose and 539 2.3-fold the Lentikats® compared to the soluble enzymes. 540 The P450 BM3 wild type served as a model in the immobilization studies in this work. On the 541 other hand, mutated P450 BM3s, usually required for oxidative reactions of non-natural 542 substrates which entail high uncoupling effects, can follow the exact same principles reported 543 here since the surface of the enzyme is usually unaffected [1], [5], [6], [45]. In this sense, 544 immobilization can be an added value to the process, for these new enzymes, due to the many 545 advantages that it confers. 546 547 Acknowledgements 548 The research for this work has received funding from the European Union project ROBOX 549 (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and Innovation 550 actions H2020-LEIT BIO-2014-1. This document reflects only the author's view and the Agency 551 is not responsible for any use that may be made of the information it contains. The 552 Department of Chemical, Biological and Environmental Engineering of Universitat Autònoma 553 de Barcelona constitutes the Biochemical Engineering Unit of the Reference Network in 554 Biotechnology and the research group 2017 SGR 1462, Generalitat de Catalunya. Authors also 555 thank COST Action CM 1303–Systems Biocatalysis for financial support and Purolite Life 556 Sciences® for the supports given. Jordi Solé acknowledges also UAB for funding his Ph.D. grant. 557 558 References 559 [1] V. B. Urlacher and M. Girhard, “Cytochrome P450 monooxygenases: An update on 24 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 560 perspectives for synthetic application,” Trends Biotechnol., vol. 30, no. 1, pp. 26–36, 561 2012. 562 [2] F. P. Guengerich, “Common and Uncommon Cytochrome P450 Reactions Related to 563 Metabolism and Chemical Toxicity,” Chem. Res. Toxicol., vol. 14, no. 6, pp. 611–650, 564 2001. 565 [3] L. O. Narhi and A. J. Fulco, “Characterization of a catalytically self-sufficient 119,000- 566 dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus 567 megaterium,” J. Biol. Chem., vol. 261, no. 16, pp. 7160–7169, 1986. 568 [4] A. J. Warman, O. Roitel, R. Neeli, H. M. Girvan, H. E. Seward, S. A. Murray, K. J. McLean, 569 M. G. Joyce, H. Toogood, R. A. Holt, D. Leys, N. S. Scrutton, and A. W. Munro, 570 “Flavocytochrome P450 BM3: an update on structure and mechanism of a 571 biotechnologically important enzyme,” Biochem. Soc. Trans., vol. 33, no. 4, pp. 747– 572 753, 2005. 573 [5] C. J. C. Whitehouse, S. G. Bell, and L.-L. Wong, “P450 BM3 (CYP102A1): connecting the 574 dots,” Chem. Soc. Rev., vol. 41, no. 3, pp. 1218–1260, 2012. 575 [6] R. Bernhardt and V. B. Urlacher, “Cytochromes P450 as promising catalysts for 576 biotechnological application: Chances and limitations,” Appl. Microbiol. Biotechnol., vol. 577 98, no. 14, pp. 6185–6203, 2014. 578 [7] C. J. Paddon, P. J. Westfall, D. J. Pitera, K. Benjamin, K. Fisher, D. McPhee, M. D. Leavell, 579 A. Tai, A. Main, D. Eng, D. R. Polichuk, K. H. Teoh, D. W. Reed, T. Treynor, J. Lenihan, H. 580 Jiang, M. Fleck, S. Bajad, G. Dang, D. Dengrove, D. Diola, G. Dorin, K. W. Ellens, S. Fickes, 581 J. Galazzo, S. P. Gaucher, T. Geistlinger, R. Henry, M. Hepp, T. Horning, T. Iqbal, L. Kizer, 582 B. Lieu, D. Melis, N. Moss, R. Regentin, S. Secrest, H. Tsuruta, R. Vazquez, L. F. 25 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 583 Westblade, L. Xu, M. Yu, Y. Zhang, L. Zhao, J. Lievense, P. S. Covello, J. D. Keasling, K. K. 584 Reiling, N. S. Renninger, and J. D. Newman, “High-level semi-synthetic production of the 585 potent antimalarial artemisinin,” Nature, vol. 496, no. 7446, pp. 528–532, 2013. 586 [8] I. Kaluzna, T. Schmitges, H. Straatman, D. Van Tegelen, M. Müller, M. Schürmann, and 587 D. Mink, “Enabling Selective and Sustainable P450 Oxygenation Technology. Production 588 of 4-Hydroxy-α-isophorone on Kilogram Scale,” Org. Process Res. Dev., vol. 20, no. 4, 589 pp. 814–819, 2016. 590 [9] U. Guzik, K. Hupert-kocurek, D. Wojcieszyńska “Immobilization as a Strategy for 591 Improving Enzyme Properties-Application to Oxidoreductases,” Molecules, vol.19, p. 592 8995–9018, 2014. 593 [10] W. Tischer and F. Wedekind, “Immobilized Enzymes: Methods and Applications,” 594 Biocatal. Discov. to Appl., vol. 200, pp. 95–126, 1999. 595 [11] R. DiCosimo, J. McAuliffe, A. J. Poulose, and G. Bohlmann, “Industrial use of 596 immobilized enzymes,” Chem. Soc. Rev., vol. 42, no. 15, p. 6437, 2013. 597 [12] J. C. S. D. Santos, O. Barbosa, C. Ortiz, A. Berenguer-Murcia, R. C. Rodrigues, and R. 598 Fernandez-Lafuente, “Importance of the Support Properties for Immobilization or 599 Purification of Enzymes,” ChemCatChem, vol. 7, no. 16, pp. 2413–2432, 2015. 600 [13] S. C. Maurer, H. Schulze, R. D. Schmid, and V. Urlacher, “Immobilisation of P450BM-3 601 and an NADP(+) cofactor recycling system: Towards a technical application of heme- 602 containing monooxygenases in fine chemical synthesis,” Adv. Synth. Catal., vol. 345, no. 603 6–7, pp. 802–810, 2003. 604 [14] I. Axarli, A. Prigipaki, and N. E. Labrou, “Engineering the substrate specificity of 605 cytochrome P450 CYP102A2 by directed evolution : production of an efficient enzyme 26 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 606 for bioconversion of fine chemicals,” vol. 22, pp. 81–88, 2005. 607 [15] E. Weber, D. Sirim, T. Schreiber, B. Thomas, J. Pleiss, M. Hunger, R. Gläser, and V. B. 608 Urlacher, “Immobilization of P450 BM-3 monooxygenase on mesoporous molecular 609 sieves with different pore diameters,” J. Mol. Catal. B Enzym., vol. 64, no. 1–2, pp. 29– 610 37, 2010. 611 [16] L. Zhao, G. Güven, Y. Li, and U. Schwaneberg, “First steps towards a Zn/Co(III)sep-driven 612 P450 BM3 reactor,” Appl. Microbiol. Biotechnol., vol. 91, no. 4, pp. 989–999, 2011. 613 [17] J. H. Lee, D. H. Nam, S. H. Lee, J. H. Park, S. J. Park, S. H. Lee, C. B. Park, and K. J. Jeong, 614 “New platform for cytochrome P450 reaction combining in situ immobilization on 615 biopolymer,” Bioconjug. Chem., vol. 25, no. 12, pp. 2101–2104, 2014. 616 [18] S. Zernia, F. Ott, K. Bellmann-Sickert, R. Frank, M. Klenner, H.-G. Jahnke, A. Prager, B. 617 Abel, A. Robitzki, and A. G. Beck-Sickinger, “Peptide-Mediated Specific Immobilization 618 of Catalytically Active Cytochrome P450 BM3 Variant.,” Bioconjug. Chem., vol. 27, no. 4, 619 pp. 1090–7, 2016. 620 [19] C. Y. Tan, H. Hirakawa, R. Suzuki, T. Haga, and F. Iwata, “Immobilization of a Bacterial 621 Cytochrome P450 Monooxygenase System on a Solid Support,” pp. 1–6, 2016. 622 [20] D. Holtmann, K. M. Mangold, and J. Schrader, “Entrapment of cytochrome P450 BM-3 623 in polypyrrole for electrochemically- driven biocatalysis,” Biotechnol. Lett., vol. 31, no. 624 5, pp. 765–770, 2009. 625 [21] V. E. V Ferrero, L. Andolfi, G. Di Nardo, S. J. Sadeghi, A. Fantuzzi, S. Cannistraro, and G. 626 Gilardi, “Protein and electrode engineering for the covalent immobilization of P450 627 BMP on gold,” Anal. Chem., vol. 80, no. 22, pp. 8438–8446, 2008. 628 [22] R. Neeli, O. Roitel, N. S. Scrutton, and A. W. Munro, “Switching pyridine nucleotide 27 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 629 specificity in P450 BM3: Mechanistic analysis of the W1046H and W1046A enzymes,” J. 630 Biol. Chem., vol. 280, no. 18, pp. 17634–17644, 2005. 631 [23] P. C. Cirino and F. H. Arnold, “A self-sufficient peroxide-driven hydroxylation 632 biocatalyst,” Angew. Chemie - Int. Ed., vol. 42, no. 28, pp. 3299–3301, 2003. 633 [24] A. Vidal-Limón, S. Águila, M. Ayala, C. V. Batista, and R. Vazquez-Duhalt, “Peroxidase 634 activity stabilization of cytochrome P450BM3 by rational analysis of intramolecular 635 electron transfer,” J. Inorg. Biochem., vol. 122, pp. 18–26, 2013. 636 [25] H. K. Chenault, E. S. Simon, and G. M. Whitesides, “Cofactor regeneration for enzyme- 637 catalysed synthesis,” Biotechnology and Genetic Engineering Reviews, vol. 6, no. 1. pp. 638 221–270, 1988. 639 [26] F. Hollmann, K. Hofstetter, and A. Schmid, “Non-enzymatic regeneration of 640 nicotinamide and flavin cofactors for monooxygenase catalysis,” Trends Biotechnol., 641 vol. 24, no. 4, pp. 163–171, 2006. 642 [27] W. Liu and P. Wang, “Cofactor regeneration for sustainable enzymatic biosynthesis,” 643 Biotechnol. Adv., vol. 25, no. 4, pp. 369–384, 2007. 644 [28] M. T. Lundemo and J. M. Woodley, “Guidelines for development and implementation of 645 biocatalytic P450 processes,” Appl. Microbiol. Biotechnol., vol. 99, no. 6, pp. 2465–2483, 646 2015. 647 [29] H. Schewe, B. A. Kaup, and J. Schrader, “Improvement of P450BM-3whole-cell 648 biocatalysis by integrating heterologous cofactor regeneration combining glucose 649 facilitator and dehydrogenase in E. coli,” Appl. Microbiol. Biotechnol., vol. 78, no. 1, pp. 650 55–65, 2008. 651 [30] N. Beyer, J. K. Kulig, A. Bartsch, M. A. Hayes, D. B. Janssen, and M. W. Fraaije, “P450 28 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 652 BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations,” 653 Appl. Microbiol. Biotechnol., 2016. 654 [31] Y. Lu and L. Mei, “Co-expression of P450 BM3 and glucose dehydrogenase by 655 recombinant Escherichia coli and its application in an NADPH-dependent indigo 656 production system,” J. Ind. Microbiol. Biotechnol., vol. 34, no. 3, pp. 247–253, 2007. 657 [32] L. D. Smith, N. Budgen, S. J. Bungard, M. J. Danson, and D. W. Hough, “Purification and 658 characterization of glucose dehydrogenase from the thermoacidophilic 659 archaebacterium Thermoplasma acidophilum.,” Biochem. J., vol. 261, no. 3, pp. 973–7, 660 1989. 661 [33] N. G. Munishwar, “Methods for Affinity Based Separations of Enzymes and Proteins,” p. 662 120. 663 [34] M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram 664 quantities of protein utilizing the principle of protein-dye binding,” Anal. Biochem., vol. 665 72, no. 1–2, pp. 248–254, 1976. 666 [35] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of 667 bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970. 668 [36] R. Omura, Tsuneo; Sato, “The Carbon Monoxide-biding pigment of Liver Microsomes,” 669 J. Biol. Chem., vol. 239, no. 7, pp. 2370–2378, 1964. 670 [37] F. P. Guengerich, M. V Martin, C. D. Sohl, and Q. Cheng, “Measurement of cytochrome 671 P450 and NADPH – cytochrome P450 reductase,” vol. 4, no. 9, pp. 1245–1252, 2009. 672 [38] A. Weckbecker and W. Hummel, “Glucose Dehydrogenase for the Regeneration of 673 NADPH and NADH,” Microb. Enzym. Biotransformations, vol. 17, no. 8, pp. 225–238, 674 2005. 29 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 675 [39] P. K. Srivastava and S. Singh, “Immobilization and Applications of Glucose-6-Phosphate 676 Dehydrogenase: a Review,” Prep. Biochem. Biotechnol., vol. 43, no. 4, pp. 376–384, 677 2013. 678 [40] A. W. Munro, D. G. Leys, K. J. Mclean, K. R. Marshall, T. W. B. Ost, S. Daff, C. S. Miles, S. 679 K. Chapman, D. A. Lysek, C. C. Moser, C. C. Page, L. Dutton, D. G. Leys, K. R. Marshall, C. 680 S. Miles, S. K. Chapman, C. C. Page, and L. Dutton, “P450 BM3 : the very model of a 681 modern flavocytochrome,” vol. 27, no. 5, pp. 250–257, 2002. 682 [41] G. Álvaro, R. Fernández-Lafuente, R. M. Blanco, and J. M. Guisan, “Immobilization- 683 Stabilization of Penicillin G Acylase from Escherichia Coli.” Applied Microbiology and 684 Biotechnology, pp. 181–195, 1989. 685 [42] E. Katchalski-Katzir and D. M. Kraemer, “Eupergit® C, a carrier for immobilization of 686 enzymes of industrial potential,” J. Mol. Catal. - B Enzym., vol. 10, no. 1–3, pp. 157–176, 687 2000. 688 [43] C. Mateo, J. M. Palomo, M. Fuentes, L. Betancor, V. Grazu, F. L??pez-Gallego, B. C. C. 689 Pessela, A. Hidalgo, G. Fern??ndez-Lorente, R. Fern??ndez-Lafuente, and J. M. Guis??n, 690 “Glyoxyl agarose: A fully inert and hydrophilic support for immobilization and high 691 stabilization of proteins,” Enzyme Microb. Technol., vol. 39, no. 2, pp. 274–280, 2006. 692 [44] I. I. Karuzina and A. I. Archakov, “The oxidative inactivation of cytochrome P450 in 693 monooxygenase reactions,” Free Radic. Biol. Med., vol. 16, no. 1, pp. 73–97, 1994. 694 [45] V. B. Urlacher and M. Girhard, “Cytochrome P450 monooxygenases: perspectives for 695 synthetic application,” Trends Biotechnol., vol. 24, no. 7, pp. 324–330, 2006. 696 TABLES 30 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 697 Table 1. Characterization of the cell free extract containing P450 BM3 and Glucose 698 dehydrogenase. Specific [Protein] (mg Activity Enzyme* (mg P450 BM3 active form Enzyme protein/mL (U/mg enz/mg prot) concentration† (µM) lysate) protein) P450 BM-3 2.82 0.524 44.9 45.4 GDH 3.63 0.146 - 699 700 *Data obtained from SDS-PAGE analysis 701 †Data obtained from CO difference spectrum assay 702 703 704 705 706 707 708 709 710 711 31 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 712 Table 2. Main characteristics of the matrices tested and their results regarding immobilization and retained activity of P450 BM3. The criterion chosen to 713 select a support for further studies sets a minimum for Immobilization at 90% and 25% for Retained activity. Functional Particle Immobilization Code group Matrix Interaction Pore diameter (Å) size Retained acitivty (%) Yield (%) (Linker) (µm) ECR8204F Epoxy Methacrylate Covalent 300-600 150-300 92 0 ECR8215F Epoxy Methacrylate Covalent 1200-1800 150-300 99 2 Amino ECR8309F Methacrylate Ionic/Covalent 600-1200 150-300 95 3 (C2) Amino ECR8315F Methacrylate Ionic/Covalent 1200-1800 150-300 97 4 (C2) Amino ECR8409F Methacrylate Ionic/Covalent 600-1200 150-300 100 6 (C6) Amino ECR8415F Methacrylate Ionic/Covalent 1200-1800 150-300 98 18 (C6) ECR8285 Epoxy (C4) Methacrylate Ionic/Covalent 400-600 300-710 100 3 None ECR8806F Methacrylate Hydrophobic 500-700 150-300 93 0 (C18) ECR1061M None Styrene/Methacrylic Hydrophobic 600-750 300-710 21 0 ECR1030M None Styrene/Methacrylic Hydrophobic 200-300 300-710 33 7 Praesto 45 Epoxy Agarose Covalent ND 45 92 63 Praesto 65 Epoxy Agarose Covalent ND 65 95 68 Praesto 90 Epoxy Agarose Covalent ND 90 95 60 32 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 714 FIGURES 715 716 Figure 1. P450 BM3 immobilization onto: A) Praesto 45 [discontinued line (-----)], 65 [dotted 717 line (·····)] and 90 [combined dotted and discontinued line (-··-··-)] [Sodium Phosphate Buffer 718 1M pH 8] B) Epoxy-agarose-Sigma [Sodium Phosphate Buffer 1M pH 8]; (I) the enzyme is 719 bound to the support (II) β-mercaptoethanol 0.2M final concentration is added to eliminate 720 the unreacted epoxy groups, C) Epoxy-agarose-UAB [Sodium Phosphate Buffer 1M pH 8] and 721 D) AMINO-agarose [Sodium phosphate buffer 0,05M pH 6]; (I) adsorption of the enzyme to the 722 support (II) incubation with certain amount of carbodiimide (EDC) (III) incubation with 0.5M 723 NaCl and desorption of the unattached protein. The activity offered always ranged from 3.5 to 724 6 UA/g of resin. The activities of a blank (squares), the supernatant (rhombus) and the 725 suspension (triangle) were continuously analysed. Error bars correspond to standard deviation 726 (n=2). 727 33 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 728 729 Figure 2. Optimization of the EDC concentration in AMINO-agarose experiments using high 730 loads. The immobilization was carried out using sodium phosphate buffer 50mM pH 6 and 731 offering 92UA/g of resin. Retained activity (black dots) and immobilization yield (grey bars) are 732 represented. Error bars correspond to standard deviation (n=2). 733 734 735 736 737 738 739 740 741 742 34 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 743 744 35 This is the pre-peer reviewed version of the following article: Solé Ferré, Jordi et al. “Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst” in Journal of Chemical Technology and Biotechnology, vol. 93 issue 7 (July 2018), which has been published in final form at DOI 10.1002/jctb.5770. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 745 Figure 3. Reaction cycles using both P450 BM3 and GDH immobilized onto: A) Epoxy-agarose- 746 UAB (10mg of agarose/mL), B) AMINO-agarose (10mg of agarose/mL) and C) Lentikats® (50mg 747 of Lentikats/mL). Reactions were carried on in 5mL scale adding 0.18-0.2UA of total activity 748 and using sodium phosphate buffer 50mM pH 7.5, 1.3mM sodium laurate, 0.2mM NADP+, 749 12.5mM D-glucose, temperature control at 25°C and constant 1000rpm agitation using a 750 Multi-Therm H5000-HC-E thermos shaker. Error bars correspond to standard deviation (n=2) 36