Agarose Vs. Methacrylate As Material Supports for Enzyme Immobilization and Continuous Processing

catalysts

Article Agarose vs. Methacrylate as Material Supports for Enzyme Immobilization and Continuous Processing

Ana I. Benítez-Mateos 1 and Martina L. Contente 2,3,*

1 Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland; [email protected] 2 School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, UK 3 Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milan, Via Celoria 2, 20133 Milan, Italy * Correspondence: [email protected]

Abstract: Enzyme immobilization has become a key strategy to improve the stability and recycling of biocatalysts, resulting in greener and more cost-efficient processes. The design of the immobilized catalysts is often focused only on the immobilization strategy, the binding chemistry between the enzyme and the support, while less attention has been paid to the physico-chemical properties of material supports. Selecting the best carrier for a specific application may greatly influence the performance of the biocatalytic reaction. Herein, we present a comparative study between the two most used material supports for protein immobilization, agarose and methacrylate. Hydrophilic agarose microbeads ensure higher retained enzymatic activity and better catalyst performance when hydrophobic compounds are involved in the biotransformation. Due to the high stickiness, lipophilic

molecules represent a major limitation for methacrylate carriers. O2-dependent reactions, in contrast, must be carried out by immobilized enzymes on methacrylate supports due to the low mechanical

Citation: Benítez-Mateos, A.I.; stability of agarose under dehydration conditions. All these parameters were tested with a special Contente, M.L. Agarose vs. focus on continuous-flow applications. Methacrylate as Material Supports for Enzyme Immobilization and Keywords: enzyme immobilization; flow biocatalysis; agarose beads; methacrylate resins Continuous Processing. Catalysts 2021, 11, 814. https://doi.org/ 10.3390/catal11070814 1. Introduction Academic Editor: Aniello Costantini Forty years ago, protein immobilization emerged as a technique to both increase the stability of enzymes and allow for their reuse and recycling. Since then, the incorporation Received: 27 May 2021 of enzymes into solid matrices has been applied for different purposes: several types of Accepted: 1 July 2021 biomaterials have been assembled by using proteins and different carriers in the field of Published: 2 July 2021 light energy conversion (e.g., photovoltaics), biosensing (e.g., detection of pesticides or heavy metals), integrated optical devices (e.g., optical switches, micro-imaging systems, Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in telecommunications technologies) as well as chemical reactions [1]. published maps and institutional affil- In particular, efficient immobilization strategies have allowed for the development of iations. heterogeneous biocatalysts with retained activity for longer periods, their easy separation from the reaction media once the reaction is completed, and even incorporation into continuous-flow reactors for process intensification [2–4]. These advances have contributed to the integration of enzymes in chemical manufac- turing processes, operating under harsh reaction conditions that are often very far from Copyright: © 2021 by the authors. their native environments [5–8]. Licensee MDPI, Basel, Switzerland. This article is an open access article The development of a “perfect biocatalyst” requires the combination of different areas distributed under the terms and of expertise such as molecular biology, protein engineering, material science, biophysics, conditions of the Creative Commons biocatalysis and chemical engineering. From the material perspective, enzymes can be Attribution (CC BY) license (https:// attached to solid supports, entrapped into gels or encapsulated in vesicles [4,9]. Enzyme creativecommons.org/licenses/by/ immobilization can also be performed by crosslinking the protein molecules, creating 4.0/). aggregates (CLEAs; crosslinked enzyme aggregates) that do not require the presence of any

Catalysts 2021, 11, 814. https://doi.org/10.3390/catal11070814 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, 814 2 of 10

support. However, their use in industrial processes is not widely adopted due to the lower activity and stability of CLEAs compared to material-based immobilization strategies [10]. Among the immobilization strategies involving a material, the attachment of enzymes to a prefabricated support is noteworthy for the easy preparation of the immobilized biocatalyst [8]. Different methodologies involving both physical and chemical approaches have been reported. Among the chemical ones, particular attention has been paid to a covalent bond between the matrix and the catalyst, especially to increase the enzyme operational stability, the easy incorporation in flow chemistry reactors and to avoid catalyst leaching when high flow rates are necessary for fast reactions [11]. Moreover, the prefabricated supports are frequently commercially available in a ready-to-use format. Typically, different material supports (agarose, methacrylate, silica, zeolites, chitosan, etc.) with various particle sizes, porosity and functional groups are tested for the immobilization of an enzyme of interest [4]. However, most of the process optimization for enzyme immobilization has been carried out by trial-and-error approaches to date, while rational immobilization procedures or predictive tools are still scarcely applied. In this work, we present a comparative study between the most widely used prefabricated material supports for purified enzyme immobilization, agarose and methacrylate microbeads. The advantages and drawbacks of each material have been analyzed and discussed, based also on previous works. The key parameters for the assessment of immobilized biocatalysts, such as retained enzyme activity after immobilization, stability in the presence of co-solvents, stability against temperature, cross-reactivity with substrates/products, and performance in packed-bed flow reactors (PBRs), are the criteria used to evaluate each material support.

2. Results and Discussion 2.1. Comparison of Enzyme Activity upon Immobilization The retained activity of 11 enzymes after immobilization on either agarose or methacrylate supports is compared in Table1, also including recently reported studies. Noteworthy, the immobilization procedures were carried out using the same binding chemistry on both carriers for the generation of a covalent bond between the biocatalysts and the support. Gs- Lys6DH (lysine-6-dehydrogenase from Geobacilus stearothermophilus), He-P5C (pyrrolidine- 5-carboxylate reductase from Halomonas elongata), HeWT (S-selective ω-transaminase from Halomonas elongata), He-AlaDH (alanine dehydrogenase from Halomonas elongata), and Ts-RTA (R-selective transaminase from Thermomyces stellatus) were immobilized, employing the epoxy/Co2+-strategy, while HRP (horseradish peroxidase), Tt-NOX (NADPH-oxidase from Thermus thermophilus), MsAcT (acyl transfersase from Mycobacterium smegmatis), HOR (β-glycosidase from Halothermothrix orenii), GalOx (galactose oxidase from Dactylium dendroides) and KRED1-Pglu (NADPH-dependent benzyl reductase from Pichia glucozyme) were immobilized on glyoxyl supports. Independently on the enzyme and the binding protocol, the biocatalysts immobilized on agarose showed a higher retained activity in all the cases (Table1). Whereas the particle size (50–150 µm for agarose beads; 100–300 µm for methacrylate beads) and the porosity (10–200 nm) of both materials are similar, the chemical nature differs. Agarose is made of sugars and is therefore more hydrophilic compared to methacrylate, which is formed by acrylic polymers. These different physico- chemical properties are plausibly the reason why immobilized enzymes on agarose show an increased retained activity [12,13]. During the enzyme immobilization on methacrylate supports, the biocatalysts are ﬁrst attracted to the carrier by hydrophobic interactions, which can provoke unfavorable rearrangements of the protein structure. Moreover, the hydrophilic nature of agarose may enhance the intraparticle mass transport of substrates and products, improving the overall performance of the immobilized enzyme [12,14]. Catalysts 2021, 11, 814 3 of 10

Table 1. Enzyme recovered activities after immobilization.

Enzyme Material Support Recovered Activity 1 (%) Results Source Agarose 91 Gs-Lys6DH Ref. [15] Methacrylate 63 Agarose 10 He-P5C Ref. [15] Methacrylate <5 Agarose 60 This work HeWT Methacrylate 30 Ref. [16] Agarose 42 He-AlaDH Ref. [17] Methacrylate 19 Agarose 95 TsRTA This work Methacrylate 67 Agarose 48 HRP This work Methacrylate 27 Agarose 55 Tt-NOX Ref. [12] Methacrylate 24 Agarose 78 MsAcT This work Methacrylate 35 Agarose 62 HOR This work Methacrylate 30 Agarose 100 GalOx This work Methacrylate 98 Agarose 35 KRED1-Pglu This work Methacrylate 16 1 Recovered activity = (speciﬁc activity of immobilized enzyme (U/mg)/speciﬁc activity of free enzyme (U/mg)) × 100. The protein loading was the same for the enzyme regardless of the material support: Gs-Lys6DH (2 mg/g), He-P5C (2 mg/g), HeWT (5 mg/g), He-AlaDH (0.5 mg/g), Ts-RTA (5 mg/g), HRP (1 mg/g), Tt-NOX (1 mg/g), MsAcT (1 mg/g), HOR (1 mg/g), GalOx (0.1 mg/g), KRED1-Pglu (10 mg/g).

Noteworthy, a tendency was observed between the recovered activities after immobilization on agarose and methacrylate. Enzymes immobilized on agarose supports were on average 2-fold more active than when immobilized on methacrylate materials. Such a difference should be taken into consideration when high enzyme activities are required for the biocatalyst application. As an exception, GalOx showed similar recovered activities regardless of the material support. Due to the low protein loading (0.1 mg/gsupport) and the high activity of its free counterpart (500–1500 U/mgenzyme as reported by the supplier), the difference in the chemical nature of the material may not have an inﬂuence at such a scale [12]. In addition, the cost of these commercial supports should be considered for the development of efﬁcient and cost-effective immobilized biocatalysts. For example, the price of methacrylate materials is between 250 and 1800 EUR/Kg, while the cost of agarose microbeads is around 1000–2000 EUR/Kg.

2.2. Stablity of the Immobilized Enzymes in Non-Natural Conditions In order to incorporate enzymes into industrial processes, the biocatalyst preparation has to be resistant to harsh conditions, which typically include high temperatures, the presence of co-solvents to enhance the substrate solubility in aqueous media, among others. Enzyme immobilization has proven to be an excellent strategy to increase the protein stability under such conditions [8,18,19]. Temperature is a key parameter for any bioprocess, but is especially relevant for biocatalytic reactions, as enzymes are complex catalysts whose industrial potential relies on their operational stability. Many studies have been performed to decrease the temperature- induced inhibition of enzymes by immobilization, thus comparing the immobilized catalyst Catalysts 2021, 11, x FOR PEER REVIEW 4 of 11

others. Enzyme immobilization has proven to be an excellent strategy to increase the protein stability under such conditions [8,18,19]. Temperature is a key parameter for any bioprocess, but is especially relevant for biocatalytic reactions, as enzymes are complex catalysts whose industrial potential relies on their operational stability. Many studies have been performed to decrease the temperature-induced inhibition of enzymes by immobilization, thus comparing the immobi- Catalysts 2021, 11, 814 lized catalyst with its free counterpart [19]. In terms of binding chemistry4 ofbetween 10 the enzyme and the support, a multipoint covalent attachment is the best strategy to promote the stability of the biocatalyst at moderately high temperatures due to the induced rigid- ificationwith of its the free protein counterpart structure [19]. In [ terms20]. Herein, of binding we chemistry have tested between the thestability enzyme at and30 °C, 37 °C and the45 support, °C of a multipoint the immobilized covalent attachment transaminase is the best strategy(imm-He to promoteWT) either the stability on agarose (HeWTof the-agarose) biocatalyst or ata moderatelymethacrylate high support temperatures (HeWT due-methacrylate) to the induced rigidiﬁcation to decipher of the effect the protein structure [20]. Herein, we have tested the stability at 30 ◦C, 37 ◦C and 45 ◦C of theof thematerial immobilized on the transaminase thermal stability (imm-HeWT) of the either immobilized on agarose enzyme (HeWT-agarose). As reported or a in the thermalmethacrylate inactivation support curves (HeWT-methacrylate) (Figure 1A), n too significant decipher the difference effect of the has material been on observed the be- tweenthermal the h stabilityalf-life of of theHeWT immobilized-agarose enzyme.and HeWT As reported-methacrylate in the thermal even at inactivation the highest tested temperaturecurves (Figure (45 1°C).A), noThese signiﬁcant results difference indicate has that been the observed thermal between stability the half-lifeis an intrinsic of pa- ◦ rameterHeWT-agarose, directly related and HeWT-methacrylate to the enzyme evenand atthe the binding highest testedchemistry temperature rather (45 thanC). the mate- These results indicate that the thermal stability is an intrinsic parameter, directly related to rial support.the enzyme and the binding chemistry rather than the material support.

A B Temperature stability Stability in 20% DMSO

100 100 Agarose Methacrylate 80 80

60 60 Agarose 30ºC 40 Methacrylate 30ºC 40

Agarose 37ºC Relative activity (%) activity Relative

Relative activity (%) activity Relative Methacrylate 37ºC 20 20 Agarose 45ºC Methacrylate 45ºC 0 0 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 Time (h) Time (h)

Figure 1. Stability of imm-HeWT at (A) different temperatures and (B) 20% v/v DMSO. The relative activity (%) was Figure 1.calculated Stability byof comparisonimm-HeWT with at the (A immobilized) different temperatures activity (U/g) at and the starting (B) 20% point. v/v TheDMSO. 100% activityThe relative of the transaminaseactivity (%) was calculated byimmobilized comparison on agarose with the corresponds immobilized to 5.7 U/g, activity while (U/g) the one at immobilized the starting on methacrylatepoint. The was100% 3.0 activity U/g. The of comparison the transaminase immobilizedof the on stability agarose of the corresp imm-HeWTonds andto 5.7 the U/g, free HeWTwhile inthe 20% one DMSO immobilized was previously on methacrylate reported [16]. was 3.0 U/g. The comparison of the stability of the imm-HeWT andDimethyl the free sulfoxideHeWT in (DMSO)20% DMSO is one was of the previously most commonly reported used[16] organic co-solvents in water-based biocatalytic reactions. Studies concerning the imm-HeWT in the presence of differentDimethyl concentrations sulfoxide (10–20%(DMSO)v/ isv) one of various of the water-miscible most common solventsly used (DMSO, organic ethanol, co-solvents in water1-propanol,-based 2-propanol, biocatalytic acetonitrile, reactions. and Studies methanol) concerning have demonstrated the imm a better-HeWT enzymatic in the presence of differentstability andconcentrations performance using(10–20% DMSO v/v [)16 of], makingvarious it water the selected-miscible co-solvent solvents in all (DMSO, the eth- reported experiments [15–17,21–25]. For this reason, the potential effect of the two matrices anol,on 1 the-propanol, imm-HeWT 2-propanol, stability in the acetonitrile, presence of 20% and DMSO methanol) was compared have demonstrated(Figure1B). The a better enzymaticdifferences stability in the residualand performance activity between using the DMSO biocatalyst [16 immobilized], making it either the selected on agarose co-solvent in allor the methacrylate reported wereexperiments barely noticeable [15-17, over21–25 a] period. For this of 70 reason, h, although the HeWT-agarosepotential effect of the two maintainedmatrices on more the than imm 90%-HeWT of its initial stability activity in duringthe presence the ﬁrst 40of h.20% In a DMSO previous w study,as compared (Figurea higher 1B). co-solventThe differences resistance in of the imm-HeWT residual versus activity the between free biocatalyst the biocatalyst has already beenimmobilized demonstrated [16]. either onAs agarose reported or for methacryl the temperatureate were effects, barely thesolvent noticeable resistance over also a period relies mainly of 70 onh, although HeWTthe-agarose enzymatic maintained architecture andmore the than binding 90% chemistry of its initial rather thanactivity the carrier. during Remarkably, the first 40 h. In a previousthe residual study activity, a higher of HeWT-agarose co-solvent resistance after 72 h inof 20% immv/-vHDMSOeWT versus was 3.5 the U/g, free while biocatalyst has alreadyHeWT-methacrylate been demonstrated preserved 2 U/g.[16]. These differences can be the consequence of a higher initialAs reported recovered for activity the temperature of the enzyme immobilizedeffects, the onsolvent agarose resistance support. also relies mainly on the enzym2.3. Stickinessatic architecture of the Susbtrate/Product and the tobinding the More Hydrophobicchemistry Supportrather than the carrier. Remarka- bly, the residualMethacrylate activity microbeads of HeWT are- moreagarose hydrophobic after 72 h when in 20% compared v/v DMSO to agarose. was 3.5 This U/g, while physico-chemical difference may affect the inertness of the carrier, especially if in close contact with apolar substrates/products. Indeed, we have experienced strong hydrophobic

Catalysts 2021, 11, x FOR PEER REVIEW 5 of 11

HeWT-methacrylate preserved 2 U/g. These differences can be the consequence of a higher initial recovered activity of the enzyme immobilized on agarose support.

2.3. Stickiness of the Susbtrate/Product to the More Hydrophobic Support Methacrylate microbeads are more hydrophobic when compared to agarose. This physico-chemical difference may affect the inertness of the carrier, especially if in close Catalysts 2021, 11, 814 contact with apolar substrates/products. Indeed, we have experienced strong5 of 10hydropho- bic interactions between aromatic compounds and the methacrylate support, even in flow conditions where no substrate/product accumulation phenomena should be ob- interactionsserved due between to the aromatic optimization compounds of and the the residence methacrylate time support, [23]. evenIn this in ﬂow experiment, conditionsHeWT-agarose where noor substrate/productHeWT-methacrylate accumulation was integ phenomenarated in a packed should- bebed observed reactor (PBR) to dueperf toor them optimization a model deamination of the residence reaction time [23 by]. using In this experiment,pyruvate and HeWT-agarose S-methylbenzylamine or HeWT-methacrylate was integrated in a packed-bed reactor (PBR) to perform a model (S-MBA) as starting materials. The reactor was operated as previously described [16]. The deamination reaction by using pyruvate and S-methylbenzylamine (S-MBA) as starting materials.consumption The reactor of S-MBA was operated as well as as previously the formation described of acetophenone [16]. The consumption were monitored of by S-MBAHPLC. as In well both as thethe formationbioreactors, of acetophenone full conversion were of monitored the substrate by HPLC. was Inachieved both the, since no bioreactors,S-MBA was full detected conversion in of the the flow substrate-through. was achieved, However, since less no thanS-MBA 50% was of detected the product in (ace- thetophenone) ﬂow-through. was However, collected less in than the 50% case of of the H producteWT-methacrylate (acetophenone) after was 10 collected column in volumes, thewhile case of 100% HeWT-methacrylate of product formation after 10 column was detected volumes, while when 100% operating of product with formation HeWT-agarose was(Figure detected 2). when operating with HeWT-agarose (Figure2).

Step 2

Step 1

Methacrylate Agarose 100

40 Conversion Conversion (%)

0 1 2 3 4 5 6 7 8 9 10 Column volumes

Figure 2. Continuous-ﬂow amination by immobilized HeWT on agarose/methacrylate supports. At Figure 2. Continuous-flow amination by immobilized HeWT on agarose/methacrylate supports. At the top, a scheme of the top, a scheme of the ﬂow system setup. Step 1: biotransformation reaction; step 2: desorption with the flow system setup. Step 1: biotransformation reaction; step 2: desorption with toluene. At the bottom, the operational performance of the PBRs. toluene.The sub Atstrate the bottom,solution the contained operational 5 mM performance S-MBA, of10 the mM PBRs. pyruvate The substrate and 0.1 solution mM PLP contained. Flow rate: 1.2 mL/min. Residence time5 mM: 1 min.S-MBA, 10 mM pyruvate and 0.1 mM PLP. Flow rate: 1.2 mL/min. Residence time: 1 min. In order to understand whether acetophenone was stuck to the methacrylate support by hydrophobicIn order to interactions, understand an whether inlet with acetophenone an apolar solvent was (toluene) stuck to was the introduced methacrylate sup- beforeport theby reactor hydrophobic to recover interactions, the product [an22 ]. inlet After with running an tolueneapolar for solvent 20 min, (toluene) 67% of the was intro- previouslyduced before stuck the acetophenone reactor to wasrecover recovered the product (Table2). [2 However,2]. After a running considerable toluene part offor 20 min, the67% product of the generated previously by the stuck bioreactor acetophenone remained trappedwas recovered in the methacrylate (Table 2). support. However, The a consid- sameerable procedure part of wasthe appliedproduct to generated the bioreactor by the with bioreactor HeWT-agarose remained to completely trapped remove in the methac- therylate traces support. of acetophenone. The same These procedur resultse was are of applied great importance to the bioreactor because thewith conversion HeWT-agarose to of the biocatalyst could be underestimated due to the stickiness of apolar compounds to completely remove the traces of acetophenone. These results are of great importance methacrylate supports. Moreover, it needs to be considered that the use of hydrophobic carriers associated with hydrophobic starting materials could increase time and costs while also reducing the greenness of the process.

Catalysts 2021, 11, x FOR PEER REVIEW 6 of 11

because the conversion of the biocatalyst could be underestimated due to the stickiness of apolar compounds to methacrylate supports. Moreover, it needs to be considered that the use of hydrophobic carriers associated with hydrophobic starting materials could increase time and costs while also reducing the greenness of the process.

Catalysts 2021, 11, 814 Table 2. In-flow recovery of acetophenone from the PBR. 6 of 10

Product Material Support % mg Table 2. In-flow recovery of acetophenone from the PBR. Agarose 4 0.2 Trapped on support 1 Methacrylate 66 Product 4.7 Material Support Agarose 100 % mg0.2 Released with toluene 2 MethacrylateAgarose 67 4 0.23.2 Trapped on support 1 1 The acetophenone trapped into the supportMethacrylate was calculated over 10 66 column volumes 4.7 considering full conversion (59.8 mM acetophenone). 2 TheAgarose acetophenone released 100 by running toluene 0.2 was Released with toluene 2 calculated considering the product previouslyMethacrylate trapped as the 100%. 67 3.2 1 The acetophenone trapped into the support was calculated over 10 column volumes considering full conversion (59.8 mM acetophenone). 2 The acetophenone released by running toluene was calculated considering the product 2.4.previously Integration trapped of O as2 the-Dependent 100%. Reactions into Flow Reactors The oxygen supply, essential for the oxidation reaction to take place, was ensured by a segmented2.4. Integration air– ofliquid O2-Dependent flow stream Reactions formed into Flow before Reactors the column containing the immobilized GalOxThe oxygen (Figure supply, 3). Air essential was delivered for the oxidationat 20 psi; reactionits flow to was take measure place, wasd as ensured previously desbycribed a segmented [26]. To air–liquidensure a flowconstant stream flow, formed a BRP before (40 thepsi column) was applied containing before the immobi-the air tank. An lized aqueous GalOx solution (Figure3 ). of Air galactose was delivered (50 mM at 20) psi; was its pumped flow was, measuredjoining the as previously airflow at the described [26]. To ensure a constant flow, a BRP (40 psi) was applied before the air tank. An T-junction, before entering the column in which the oxidation reaction occurs in ap- aqueous solution of galactose (50 mM) was pumped, joining the airflow at the T-junction, proximatelybefore entering 10 min. the columnA BPR in (5 which psi) theensured oxidation a constant reaction occurs and controlled in approximately flow of 10 min. aqueous phaseA BPR leaving (5 psi) the ensured column. a constant The aqueous and controlled stream flow was of collected aqueous phaseand the leaving unreacted the column. galactose wasThe me aqueousasured streamusing a was commercial collected andly available the unreacted kit ( galactoseR-biopharm was) measured. Although using no adifference com- in themercially reaction available performance kit (R-biopharm). could be Although observed no in difference terms of in molar the reaction conversion performance at the be- ginningcould of be the observed process in (80% terms m.c. of molar methacrylate conversion support; at the beginning 76% m.c. of agarose the process carrier), (80% m.c. a better operationmethacrylateal stability support; was 76% obtained m.c. agarose using carrier), the methacrylate a better operational material stability over was time. obtained After 48 h of continuoususing the methacrylate processing material, an increas over time.e in Afterpressure 48 h ofin continuous addition to processing, dehydration an increase problems in pressure in addition to dehydration problems was observed in the case of the agarose was observed in the case of the agarose GalOx, giving rise to lower productivity (60% GalOx, giving rise to lower productivity (60% m.c. after 48 h) and lower enzymatic effi- m.c. after 48 h) and lower enzymatic efficiency. Constant delivery of the desired aldehyde ciency. Constant delivery of the desired aldehyde was observed for the methacrylate GalOx was(78% observed mc after for 48 the h). methacrylate GalOx (78% mc after 48 h).

FigureFigure 3. Continuous 3. Continuous-ﬂow-flow scheme scheme of ofthe the D D-galactose-galactose oxidationoxidation through through immobilized immobilized GalOx GalOx on either on agaroseeither agarose or methacry- or meth- acrylatelate supports. supports. 3. Materials and Methods 3. Materials and Methods 3.1. Materials 3.1. MaterialsGalactose oxidase from D. dendroides (GalOx), peroxidase from horseradish (HRP), 0 AzBTS-(NHGalactose4 oxidase)2, S-Methylbenzylamine, from D. dendroidesR-Methylbenzylamine, (GalOx), peroxidase pyruvate, from horseradish pyridoxal-5 (HRP),- AzBTSphosphate,-(NH4) D-galactose,2, S-Methylbenzylamine,p-nitrophenyl acetate, R-Methylbenzylamine,p-nitrophenyl-β-D-glucopyranoside, pyruvate, Benzil,pyridoxal-5NADPH′-phosphate, were purchasedD-galactose from, p- Sigmanitrophenyl Aldrich acetate, Gillingham, p-nitrophenyl U.K. Plain agarose-β-D-glucopyranoside, (6BCL) was purchased from Agarose Bead Technologies (ABT), Madrid, Spain. The methacrylate supports were donated by Resindion S.R.L. and Purolite®. All other reagents were of analytical grade.

Catalysts 2021, 11, 814 7 of 10

3.2. Protein Expression and Purification Protein expression and purification were carried out as previously described: Ms- AcT [27]; HOR [28]; TsRTA [29]; HeWT [30]; KRED1-Pglu [22]. HeWT, TsRTA, MsAcT as pure proteins were stored at 4 ◦C, HOR at room temperature and KRED1-Pglu at −20 ◦C. Protein quantification was performed by Bradford assay [13].

3.3. Enzymatic Activity Assays Activity assays were carried out in 96-well plates for both the free and immobilized enzymes. An adapted version of previous protocols [29,30] was used for TsRTA and HeWT. Briefly, 200 µL of a reaction mixture containing 2.5 mM R- or S-MBA, 2.5 mM pyruvate and in 50 mM phosphate buffer at pH 8.0 with 2.5% DMSO was prepared. The reaction was triggered by adding 5 µL of the free enzyme (0.5 mg/mL) or immobilized one (suspension of 1:5 w/v). The increase in UV absorbance was monitored at 245 nm for 5 min at 30 ◦C. Free TsRTA specific activity 2.6 U/mg, free HeWT 1.9 U/mg. HRP activity: 200 µL of a reaction mixture containing 2.5 mM D-galactose, 2.5 mM AZBTS, 0.02 mg/mL GalOx in 50 mM phosphate buffer at pH 7.5 was prepared. The reaction was triggered by adding 5 µL of free HRP or immobilized HRP (suspension of 1:5 w/v). The increase in absorbance was monitored at 420 nm for 10 min at 25 ◦C. Free enzyme specific activity: 150 U/mg. An adapted version of previous protocols [27,28] was used for MsAcT and HOR. Briefly, 200 µL of a reaction mixture containing 0.56 mM para-nitrophenyl acetate, 0.1% v/v EtOH, or 10 mM para-nitrophenyl-β-D-glucopyranoside (pNPG) in 100 mM phosphate buffer at pH 8.0 or 50 mM HEPES buffer at pH 7.4 was prepared. The reaction was triggered by adding 2 µL of free MsAcT dil 1:100 (0.0005 mg/mL) or immobilized (suspension of 1:10 w/v) or 2 µL of free HOR dil 1:10 (0.003 mg/mL) or immobilized suspension (1:5 w/v). The increase in absorbance was monitored at 400 nm for 5 min at 25 ◦C. Free MsAcT specific activity: 180 U/mg, free HOR: 7 U/mg GalOx activity: 200 µL of a reaction mixture containing 2.5 mM D-galactose, 2.5 mM AZBTS, 0.0025 mg/mL HRP in 50 mM phosphate buffer at pH 7.5 was prepared. The reaction was triggered by adding 5 µL of free GalOx or immobilized GalOx (suspension of 1:5 w/v). The increase in absorbance was monitored at 420 nm for 10 min at 25 ◦C. Free enzyme specific activity: 700 U/mg. KRED1-Pglu activity: An adapted version of a previous protocol was followed [22]. A total of 200 µL of a reaction mixture containing 0.25 mM NADPH, 0.5 mM Benzil, 0.1% v/v DMSO, 0.7 mg/mL KRED1-Pglu in 50 mM Tris-HCl buffer at pH 8.0 was prepared. The reaction was triggered by adding 100 µL of free or immobilized KRED1-Pglu (suspension of 1:5 w/v). The decrease in absorbance was monitored at 340 nm for 5 min at 25 ◦C. Free enzyme specific activity: 20 mU/mg.

3.4. Enzyme Immobilization on Epoxy/Co2+-Supports Epoxy-agarose microbeads were prepared as previously described [31]. Epoxy- methacrylate (EP403/S or ECR8204F) was commercially available. The addition of Co2+- chelates to either epoxy-agarose or epoxy-methacrylate was carried out by incubating 1 g of epoxy-support with 2 mL of modification buffer (0.1 M sodium borate, 2 M iminodiacetic acid pH 8.0) for 2 h. After filtration and washing steps, the resin was incubated with 5 mL of Metal Buffer (30 mg/mL of CoCl2). After filtration and washing steps, 5 mL of (6×)Histag-enzyme was added to the resin and the suspension was incubated for 6 h under shaking. The immobilized enzyme was washed with 3 mL of desorption buffer (50 mM EDTA, 0.5 M NaCl in 50 mM phosphate buffer, pH 7.2). Finally, the remaining epoxy groups were blocked by incubation with 4 mL of 3 M glycine pH 8.5 overnight and afterwards the immobilized enzyme was washed with the appropriate buffer for its storage. Catalysts 2021, 11, 814 8 of 10

3.5. Enzyme Immobilization on Glyoxyl Supports Epoxy-agarose microbeads and epoxy-methacrylate (EP403/S or ECR8204F) were activated with glyoxyl groups following a modified version of a previous protocol [32]. Briefly, 1 g of either epoxy-agarose or epoxy-methacrylate was incubated with 10 mL of 100 mM H2SO4 overnight under orbital shaking. Then, the support was filtered and washed 10 times with 10 volumes of H2O. The resulting glyceryl support was oxidized with 10 mL of 30 mM NaIO4 for 2 h under orbital shaking. Finally, the glyoxyl support was washed ◦ 10 times with H2O and stored at 4 C until use. For the enzyme immobilization, 10 mL of enzyme at the desired concentration in 100 mM sodium bicarbonate buffer at pH 10.0 was mixed with 1 g of glyoxyl support and incubated under orbital shaking for 3 h. Then, the suspension was filtered and the immobilization yield (%) was calculated by measuring the remaining activity in the flow- through. The immobilized enzyme was incubated with 20 mM of piperidine–borane to reduce the imine bonds between the enzyme and the aldehyde groups on the support. The immobilized enzyme was washed with the appropriate buffer for its storage.

3.6. Enzyme Stability Test in DMSO A total of 0.9 mL of 50 mM phosphate buffer at pH 8.0 containing 20% v/v DMSO was added to 0.1 g of immobilized HeWT (5 mg/g) on either agarose or methacrylate. The suspension was incubated at 25 ◦C under shaking for 72 h. Samples were withdrawn at different time points and the enzyme activity was measured as described in Section 3.3.

3.7. Enzyme Stability Test at 45 ◦C A total of 0.9 mL of 50 mM phosphate buffer at pH 8.0 was added to 0.1 g of immobilized HeWT (5 mg/g) on either agarose or methacrylate. The suspension was incubated in a water bath at 45 ◦C with magnetic stirring for 15 h. Samples were withdrawn at different time points and the enzyme activity was measured as described in Section 3.3.

3.8. Continuous Flow Reactions Flow biotransformations were performed using a R2S/R4 Vapourtec flow reactor equipped with a V3 pump and an Omnifit glass column (6.6 mm i.d. × 100 mm length) filled with the immobilized enzyme (1 g) as a packed-bed reactor (PBR). A first equilibration step was performed by running buffer at 1 mL/min for 10 min (50 mM phosphate buffer pH 8.0 for HeWT, pH 7.5 for GalOx). Subsequently, solutions of substrates/cofactors at different concentrations depending on the enzyme were mixed in a T-tube and pumped through the PBR containing the immobilized biocatalyst (5 mM S-MBA, 10 mM pyruvate, and 0.1 mM PLP in 50 mM phosphate buffer pH 8.0 with 2.5% v/v DMSO for HeWT, 50 mM D-galactose for GalOx phosphate buffer pH 7.5). In the case of GalOx, a segmented flow air/liquid was formed to provide the O2 necessary for the oxidation reaction. Samples were collected after each column volume and analyzed by HPLC or D-galactose commercial kit by R-biopharm, specific for the detection of D-galactose in complex matrix. For the recovery of the product stuck to the support, a first washing step was performed by running buffer at 1 mL/min for 10 min. Then, toluene was flushed at 0.59 mL/min for 20 min. Samples were collected after each column volume and analyzed by HPLC.

3.9. HPLC Analysis The products were analyzed by HPLC equipped with a C18 column. The samples were diluted in a solution of 1:1 (v/v) 0.1% HCl and acetonitrile. S-MBA (Rt. 4.6 min) and acetophenone (Rt. 6 min) were detected using a UV detector at 250 nm after a gradient method 5:95 to 95:5 (H2O:MeCN 0.1%TFA) over 4 min with a ﬂow rate of 0.8 mL/min at 45 ◦C. Standards of acetophenone were also analyzed for the calibration curve. Catalysts 2021, 11, x FOR PEER REVIEW 9 of 11

3.9. HPLC Analysis The products were analyzed by HPLC equipped with a C18 column. The samples were diluted in a solution of 1:1 (v/v) 0.1% HCl and acetonitrile. S-MBA (Rt. 4.6 min) and acetophenone (Rt. 6 min) were detected using a UV detector at 250 nm after a gradient method 5:95 to 95:5 (H2O:MeCN 0.1%TFA) over 4 min with a flow rate of 0.8 mL/min at 45 °C. Standards of acetophenone were also analyzed for the calibration curve.

Catalysts 2021, 11, 814 4. Conclusions 9 of 10 In this study, a comparison between agarose and methacrylate for enzyme immobilization has been carried out. Using the same binding chemistry and enzymatic loading 4. Conclusions for each enzyme on both of the carriers, we elucidated the role of the material support on typical biotransformationsIn this study, a comparison with betweena special agarose focus and on methacrylateflow processing for enzyme. Eleven immobi- different bio- lization has been carried out. Using the same binding chemistry and enzymatic loading catalystsfor each have enzyme been onconsidered both of the, carriers, demonstrating we elucidated that the the role physico of the material-chemical support properties of the matrixon typical may biotransformations influence the withperformance a special focus of the on flowflow processing.-reaction. Eleven While different the hydrophilic agarosebiocatalysts ensures have a 2- beenfoldconsidered, more active demonstrating catalyst than that thethe physico-chemical methacrylate counterpart properties , and a betterof performance the matrix may when influence apolar the performance aromatic compounds of the flow-reaction. are involved While the in hydrophilic the reaction, more agarose ensures a 2-fold more active catalyst than the methacrylate counterpart, and a lipophilicbetter performancemethacrylate when supports apolar aromatic present compounds greater mechanical are involved in stability the reaction,, especially more under dehydrationlipophilic conditions. methacrylate No supports influence present of greater the carrier mechanical properties stability, has especially been observed under in the enzymaticdehydration resistance conditions. to the No reaction influence parameters of the carrier, propertiessuch as temperature has been observed and inco the-solvents, as they enzymaticare intrinsic resistance characteristics to the reaction more parameters, related such to the as temperature protein structure. and co-solvents, Focusing as on the they are intrinsic characteristics more related to the protein structure. Focusing on the final final application, this report can be considered a “first-step-guide” for a more rational application, this report can be considered a “first-step-guide” for a more rational selection selectionof the of two the most two employed most employed matrices for matri proteinces immobilization,for protein immobilizatio allowing for an fast, allowing and for a fast andcost-efficient cost-efficient optimization optimization of the process of the parameters process (Figureparameters4). (Figure 4).

Agarose supports Methacrylate supports

✓ Higher recovered activity ✓ Lower cost ✓ No stickiness of the substrate/product ✓ Better performance with air-based reactions ✓ Biodegradable ✓ Better performance at higher flow-rates

Figure 4.Figure Summary 4. Summary of the of main the main advantages advantages of of agarose and and methacrylate methacrylate material material supports. supports.

AuthorAuthor Contributions: Contributions: Conceptualization,Conceptualization, A.I.B.-M. A.I.B.-M and. and M.L.C.; M.L.C. methodology,; methodology, formal analysis, formal analysis, investigation, data curation, and resources, A.I.B.-M. and M.L.C.; writing—original draft preparation, investigation,A.I.B.-M.; writing—reviewdata curation, andand editing, resources, M.L.C. A.I.B. All authors-M. and have M.L.C.; read and writing agreed— to theoriginal published draft preparation, A.I.B.version-M.; of the writing manuscript.—review and editing, M.L.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University of Bern “Seal of Excellence Fund”, grant Funding:number This SELF19-03 research BIORPHANDRUG was funded by(A.I.B.-M); the University the European of Union’s Bern “Seal Horizon of 2020 Excellence research and Fund”, grant numberinnovation SELF19 programme-03 BIORPHANDRUG under the Marie Sklodowska-Curie (A.I.B.-M); the grant European number Union’s 792804 AROMAs-FLOW Horizon 2020 research (M.L.C.). and innovation programme under the Marie Sklodowska-Curie grant number 792804 ARO- MAs-DataFLOW Availability (M.L.C.) Statement:. Not applicable. Acknowledgments: The authors are grateful to Francesca Paradisi for her support and the insightful Acknowledgments: The authors are grateful to Prof. Francesca Paradisi for her support and the discussions. We wish to thank Resindion S.R.L. and Purolite® for donating the methacrylate resins.. insightful discussions. We wish to thank Resindion S.R.L. and Purolite® for donating the methac- rylateConﬂicts resins.. of Interest: The authors declare no conﬂict of interest. References Conflicts of Interest: The authors declare no conflict of interest. 1. Nagy, L.; Magyar, M.; Szabo, T.; Hajdu, K.; Giotta, L.; Dorogi, M.; Milano, F. Photosynthetic machineries in nano-systems. Curr. Protein Pept. Sci. 2014, 15, 363–373. [CrossRef][PubMed] References2. Guisan, J.M.; López-Gallego, F.; Bolivar, J.M.; Rocha-Martín, J.; Fernandez-Lorente, G. The Science of Enzyme Immobilization. In 1. Nagy, L.; MethodsMagyar, in MolecularM.; Szabo, Biology T.;; HumanaHajdu, PressK.; Giotta, Inc.: New L. York,; Dorogi, NY, USA, M.; 2020;Milano, Volume F. 2100,Photosynthetic pp. 1–26. machineries in nano-systems. Curr. 3.Protein Bommarius, Pept. Sci. A.S.; 2014 Paye,, 15 M.F., 363 Stabilizing–373, doi biocatalysts.:10.2174/1389203715666140327102757Chem. Soc. Rev. 2013, 42, 6534–6565.. [CrossRef] 4. Sheldon, R.A.; van Pelt, S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. 2. Guisan, J.M.; López-Gallego, F.; Bolivar, J.M.; Rocha-Martín, J.; Fernandez-Lorente, G. The Science of Enzyme Immobilization. [CrossRef] In Methods5. Sheldon, in Molecular R.A.; Woodley, Biology; J.M. Humana Role of BiocatalysisPress Inc.: inNew Sustainable York, Chemistry.NY, USA,Chem. 2020; Rev. Vol2018ume, 118 2100,, 801–838. pp. 1 [–CrossRef26. ] 3. Bommarius,6. Chapman, A.S.; Paye, J.; Ismail, M.F. E.A.; Stabilizing Dinu, Z.C. biocatalysts. Industrial Applications Chem. Soc. of Enzymes: Rev. 2013 Recent, 42, Advances, 6534–6565, Techniques, doi:10.1039/c3cs60137d. and Outlooks. Catalysts 4. Sheldon, R.A.;2018, 8van, 238. Pelt, [CrossRef S. Enzyme] immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235, doi:10.1039/C3CS60075K.7. Winkler, C.K.; Schrittwieser, J.H.; Kroutil, W. Power of Biocatalysis for Organic Synthesis. ACS Cent. Sci. 2021, 7, 55–71. [CrossRef] [PubMed] 8. Basso, A.; Serban, S. Industrial applications of immobilized enzymes—A review. Mol. Catal. 2019, 479, 110607. [CrossRef]

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