Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter Sp

catalysts

Article Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent

Mayerlenis Jimenez Rojas 1,†, Murilo Amaral-Fonseca 1, Gisella Maria Zanin 2, Roberto Fernandez-Lafuente 3,* , Raquel de Lima Camargo Giordano 1 and Paulo Waldir Tardioli 1,* 1 Graduate Program in Chemical Engineering, Department of Chemical Engineering, Federal University of São Carlos, Rod. Washington Luiz, km 235, 13565-905 São Carlos, SP, Brazil; [email protected] (M.J.R.); [email protected] (M.A.-F.); [email protected] (R.d.L.C.G.) 2 Graduate Program in Chemical Engineering, Department of Chemical Engineering, State University of Maringá, Av. Colombo, 5790, Bloco D90, Jd. Universitário, 87020-900 Maringá, PR, Brazil; [email protected] 3 Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain * Correspondence: rﬂ@icp.csic.es (R.F.-L.); [email protected] (P.W.T.); Tel.: +34-915954941 (R.F.-L.); +55-16-3351-9362 (P.W.T.) † Present address: Chemical Engineering Program, Universidad del Atlántico, Carrera 30 # 8-49, Puerto Colombia, Atlántico 081001, Colombia. Received: 10 January 2019; Accepted: 22 January 2019; Published: 30 January 2019

Abstract: Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration of the crosslinker) were evaluated on the production of CLEAs of CGTase. Among the seven studied precipitants, acetone with a 75% (v/v) concentration produced the aggregates of CGTase with higher activity, which retained 97% of the initial activity. Concerning the cross-linker (glutaraldehyde, starch–aldehyde, and pectin–aldehyde), starch–aldehyde produced the most active CLEAs. The use of bovine serum albumin as co-feeder decreased the expressed activity. Addition of polyethylenimine at the end of cross-linking step prevented the leakage of the enzyme and the subsequent Schiff’s bases reduction with sodium borohydride permitted to maintain 24% of the initial activity even with the large dextrin as substrate. The optimal conditions for the immobilization process required were defined as 75% (v/v) acetone as precipitation reagent for 1 h at 20 ◦C, 20 mM starch–aldehyde as crosslinking reagent for 2 h at 20 ◦C, treatment with 1 mg/mL of polyethylenimine for 5 min, reduction with 1 mg/mL of sodium borohydride. The CLEAs of CGTase were active catalyst (similarly to the free enzyme) in the production of cyclodextrins at 50 ◦C and pH 6.0 for 6 h reaction, maintaining intact their structures. Besides this, after five cycles of 3 h the total cyclodextrin yield was 80% of the initial value (first batch, with around 45% CD yield).

Keywords: CGTase; CLEAs; immobilization; crosslinking agent effect; starch; cyclodextrins

1. Introduction Cyclodextrin glucosyltransferases (CGTase) (EC 2.4.1.19) are extracellular microbial enzymes capable of converting starch and their derivatives into cyclodextrins (CDs), which are non-reducing

Catalysts 2019, 9, 120; doi:10.3390/catal9020120 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 120 2 of 17 cyclic oligosaccharides formed commonly by 6, 7, or 8 glucose units linked by α(1→4) bonds, namely of α-, β-, and γ-CD, respectively [1,2]. Specifically, CGTase from Thermoanaerobacter sp. produces mainly α- and β-CD, and small amount of γ-CD [3]. CDs have a truncated cone shape with a hydrophobic inner cavity that allows encapsulation of hydrophobic organic molecules, increasing its solubility in some media and increasing its stability. Due to this inclusion property, CDs have many applications in the food, cosmetics, pharmaceutical, and textile industries [4]. In addition to the cyclization reaction (CD production), this enzyme also catalyzes other two intermolecular transglycosylation reactions (coupling and disproportionation) and the hydrolysis reaction of the starch [3,5]. Although it is mainly used at industrial scale for the production of cyclodextrins, recent studies performed by Yu et al. [6] have demonstrated that CGTase from Paenibacillus sp. CGMCC 5316 can be used to eliminate the bitter taste of a natural sweetener known as stevioside by a reaction of transglycosylation using starch as a glucose donor. Despite the excellent catalytic properties of the enzymes (high specificity and selectivity), their use as industrial catalysts is still limited due to their low operational stability, difficult recovery and reuse [7]. The immobilization of enzymes has been first designed to permit an easy reuse of this expensive catalyst, but it also makes their application feasible in continuous processes, allowing their separation and recycling for long periods of time if the immobilized enzyme is stable enough [7–10]. Moreover, a proper immobilization may improve many other enzyme limitations, like stability, activity, specificity or selectivity, resistance to inhibitors, even purity [11]. A versatile carrier-free immobilization technique for enzyme immobilization has attracted increasing attention [12–14]. This technique called CLEAs (cross-linked enzyme aggregates) consists in the physical formation of enzyme aggregates (induced by salts, organic solvents, non-ionic polymers, etc.) followed by chemical crosslinking with bifunctional (usually glutaraldehyde) or polyfunctional (e.g., aldehyde–dextran) agents [15]. In comparison to enzymes immobilized onto solid supports, CLEAs are cheaper biocatalysts that exhibit higher mass loadings [16,17]. However, some problems of the CLEAs have been reported, such as intraparticle diffusional limitations due to the high enzyme loading and uncontrolled pores and mild physical resistance [18]. Moreover, in some cases the crosslinking step may be problematic, when the enzyme has few Lys residues in its surface [19–23]. Diffusion limitations and crosslinking problems may be decreased if using a feeder rich in amino groups, which decreases the loading of the enzyme and facilitates the crosslinking step. This feeder may be an aminated polymer, like polyethylenimine [24], or a protein rich in amino groups (e.g., bovine serum albumin or soy protein) [23,25]. In some instances, the enzyme has been chemically aminated [26]. As crosslinker, glutaraldehyde is the most utilized molecule [27]. Other alternatives proposed in literature are polyfunctional polymers, like aldehyde–dextran [28], aldehyde–pectin [29], or aldehyde–starch [30]. Advantages of these reagents are a lower chemical modification of internal amino groups of the protein (e.g., groups related to the catalysis located in internal pockets [28]), reduction of diffusional problems by enlarging the pores [30], and decreasing enzyme loading. CLEAs technology has been successfully applied to several amylolytic enzymes [20,23,29,31,32], but, to the best of our knowledge, there are no papers related to the immobilization of CGTase using this technique. These enzymes have been immobilized and stabilized by conventional adsorption methods (using ion exchange resins, chitosan, silica, among others) or by covalent binding to solid supports such as glyoxyl agarose, Eupergit C, chitosan, etc. [33–38]. Particularly, CGTase from Thermoanaerobacter sp. has been only immobilized on solid matrices, such as glutaraldehyde-activated chitosan [38], glutaraldehyde-activated silica [33], octadecyl-Sepabeads [36], glyoxyl-activated agarose [37], glyoxyl-activated silica [36], Eupergit C [34,39], Amberlite IRA-900 [40], CNBr- and 1,6-diaminohexane-activated Sepharose [41]. In this context, this work reports the first preparation of CLEAs of CGTase from Thermoanerobacter sp., evaluating different parameters such as precipitating agent nature and its concentration, the best bifunctional or macromolecular crosslinkers and its concentration and the effect of the addition of Catalysts 20192018,, 98,, 120x FOR PEER REVIEW 3 of 17 concentration, the best bifunctional or macromolecular crosslinkers and its concentration and the some feeders (proteins or PEI). In addition, the applicability and reusability of CLEAs of CGTase were effect of the addition of some feeders (proteins or PEI). In addition, the applicability and reusability also determined. of CLEAs of CGTase were also determined. 2. Results and Discussion 2. Results and Discussion 2.1. Selection of Precipitating Agent 2.1. Selection of precipitating agent The aggregation/precipitation step of CGTase was evaluated using methanol, ethanol, tert-butanol,The aggregation/precipitation acetone, isopropanol, step acetonitrile, of CGTase and was saturated evaluated ammonium using methanol, sulfate ethanol, solution tert as- precipitants.butanol, acetone, After isopropanol, precipitation, acetonitrile, the precipitate and sa wasturated recovered ammonium by centrifugation sulfate solution and as re-dissolved precipitants. in 50After mM precipitation, sodium citrate the buffer precipitate pH 6.0. Aswas shown recovered in Figure by1 ,centrifugationtert-butanol yielded and re-dissolved the lowest precipitation in 50 mM yieldsodium (60%), citrate whereas buffer acetone pH 6.0.allowed As shown a precipitation in Figure 1, tert- yieldbutanol of 98.4%. yielded Consequently, the lowest acetone precipitation was selected yield as(60%), precipitant whereas of acetone CGTase allowed for CLEA a precipitation preparation. yield of 98.4%. Consequently, acetone was selected as precipitant of CGTase for CLEA preparation.

100

20 Precipitation yield (%)

0 l l o ol ile ol n tr no an i ta an h th Et eton Acetone rc-bu Me o-propa Ac A. sulphate e Is t

Precipitant Figure 1.1.Precipitation Precipitation of CGTaseof CGTase from Thermoanaerobacterfrom Thermoanaerobactersp. with differentsp. with precipitants. different Precipitationprecipitants. conditions:Precipitation 1 hconditions: reaction, 20 1 ◦hC reaction, at 200 rpm 20°C stirring, at 200 90% rpm (v /stirring,v) precipitant 90% (v/v) concentration, precipitant enzyme concentration, solution preparedenzyme insolution 50 mM prepared citrate buffer in pH50 6.0.mM Precipitates citrate bu wereffer recoveredpH 6.0. byPrecipitat centrifugationes were and recovered re-dissolved by forcentrifugation enzyme activity and re-dissolved measurement. for The enzyme total initialactivity enzyme measurement. activity was The taken total asinitial 100%. enzyme activity was taken as 100%. 2.2. Effect of the Precipitant Concentration on the Precipitated Activity of CGTase 2.2. EffectDifferent of the precipitant volumetric concentratio concentrationsn on the ofprecipitated acetone activity were evaluatedof CGTase in the precipitation of ThermoanaerobacterDifferent volumetricsp. CGTase. concentrations The increase of in theacet volumetricone were concentrationevaluated in ofthe acetone precipitation from 75% toof 90%Thermoanaerobacter (v/v) did not affect sp. CGTase. the activity The recoveredincrease in after the volumetr re-dissolvingic concentration the CGTase precipitate,of acetone from yielding 75% anto average90% (v/v) CGTase did not precipitation affect the activity yield aroundrecovered of 97%. after So, re-dissolving a precipitant the concentration CGTase precipitate, of 75% was yielding selected an foraverage further CGTase assays precipitation of CLEA preparation yield around (aggregation of 97%. followed So, a precipitant by crosslinking). concentration of 75% was selected for further assays of CLEA preparation (aggregation followed by crosslinking). 2.3. Effect of Glutaraldehyde Concentration and Reaction Time

2.3. EffectThe concentrationof glutaraldehyde of co glutaraldehydencentration and isreaction a parameter time that must be optimized, because an excess of glutaraldehyde can cause excessive modification of the protein. On the other hand, too low The concentration of glutaraldehyde is a parameter that must be optimized, because an excess concentrations of glutaraldehyde can promote inefficient cross-linking, leading to loss of enzyme in of glutaraldehyde can cause excessive modification of the protein. On the other hand, too low the reaction medium [42]. The objective is to modify each primary amino group of the enzyme with concentrations of glutaraldehyde can promote inefficient cross-linking, leading to loss of enzyme in just one molecule of glutaraldehyde [43]. the reaction medium [42]. The objective is to modify each primary amino group of the enzyme with Figure2A shows the global activity recoveries in the CLEAs and the activity losses in the first just one molecule of glutaraldehyde [43]. washing (in relation to the total initial activity). The concentration of 10 mM glutaraldehyde was Figure 2A shows the global activity recoveries in the CLEAs and the activity losses in the first insufficient to properly cross-link the enzyme aggregates, since all enzyme activity was lost during washing (in relation to the total initial activity). The concentration of 10 mM glutaraldehyde was the washing step (data not shown). Higher concentration of glutaraldehyde allowed cross-linking; insufficient to properly cross-link the enzyme aggregates, since all enzyme activity was lost during the washing step (data not shown). Higher concentration of glutaraldehyde allowed cross-linking;

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however, it is unable to prevent the presence of enzyme activity in the supernatant and produces CatalystsCatalysts 20182019,, 89,, x 120 FOR PEER REVIEW 44 of of 17 17 great losses of activity (more than 90%). Cross-linking with 20 mM glutaraldehyde yielded the however,highest global it is unable activity to recovery prevent (aroundthe presence 10%), of but enzyme it was activityobserved in an the activity supernatant loss above and 80%.produces Even however, it is unable to prevent the presence of enzyme activity in the supernatant and produces great greatevaluating losses theof crosslinkingactivity (more time than at this90%). glutaralde Cross-linkinghyde concentration with 20 mM (Figure glutaraldehyde 2B), the highest yielded global the losses of activity (more than 90%). Cross-linking with 20 mM glutaraldehyde yielded the highest global highestactivity global recovery activity was recoverymaintained (around around 10%), 10%. but This it waslow observedactivity recovery an activity could loss be above associated 80%. Even with activity recovery (around 10%), but it was observed an activity loss above 80%. Even evaluating the evaluatingdiffusional the problems crosslinking of the time large at this substrate glutaralde withhydein theconcentration supramolecular (Figure structure 2B), the highest of the globalCLEA crosslinking time at this glutaraldehyde concentration (Figure2B), the highest global activity recovery activity[18,30,44], recovery inadequate was maintained enzyme crosslinking around 10%. that This permitted low activity the loss recovery of a great could percentage be associated of enzyme with was maintained around 10%. This low activity recovery could be associated with diffusional problems diffusionalmolecules, problemsor inactivation of the inducedlarge substrate by glutaraldehyde, within the supramolecular or a combination, structure because of the extensive CLEA of the large substrate within the supramolecular structure of the CLEA [18,30,44], inadequate enzyme [18,30,44],crosslinking inadequate may result enzyme in a distortion crosslinking of the that enzy permittedme structure the loss [27,45]. of a great However, percentage the incubation of enzyme of crosslinking that permitted the loss of a great percentage of enzyme molecules, or inactivation induced molecules,the free enzyme or inactivation into 20 mM glutaraldehyde,induced by glutaraldehyde, prepared in 50 mMor asodium combination, citrate buffer because pH 6.0, extensive showed by glutaraldehyde, or a combination, because extensive crosslinking may result in a distortion of the crosslinkingthat glutaraldehyde may result has in a verya distortion low deleterious of the enzy effectme on structure the free enzyme[27,45]. However,activity (activity the incubation loss around of enzyme structure [27,45]. However, the incubation of the free enzyme into 20 mM glutaraldehyde, the20% free after enzyme 3 h as into shown 20 mM in Figure glutaraldehyde, 3). Thereby, prepared other strategies in 50 mM were sodium evaluated citrate bufferaiming pH to 6.0,increase showed the prepared in 50 mM sodium citrate buffer pH 6.0, showed that glutaraldehyde has a very low deleterious thatglobal glutaraldehyde recovery activity has a of very the lowCGTase deleterious CLEAs. effect on the free enzyme activity (activity loss around effect on the free enzyme activity (activity loss around 20% after 3 h as shown in Figure3). Thereby, 20% after 3 h20 as shown in Figure 3). Thereby, other strategies15 were evaluated aiming to increase the other strategies (A) were evaluated aiming to increase the global recovery(B) activity of the CGTase CLEAs. global recovery activity Global activity of the recovery CGTase in the CLEA CLEAs. Activity measured in the washing solution 12 2015 15 (A) (B) Global activity recovery in the CLEA 9 Activity measured in the washing solution 12 1510 6 9

Activity (%) 105 3 6 Global activity recovery (%)

Activity (%) 50 0 20 30 50 3 1234 Glutaraldeyde Concentration (mM) Time (h) Global activity recovery (%) 0 0 1234 Figure 2. (A) Effect20 of glutaraldehyde 30 concentration 50 on the global activity recovery of CGTase CLEAs Glutaraldeyde Concentration (mM) Time (h) (enzyme solution prepared in 50 mM sodium citrat e buffer pH 6.0, precipitation with acetone at a FigureFigurevolumetric 2. 2. ((AA )) concentrationEffect Effect of of glutaraldehyde glutaraldehyde of 75%, 1 concentration concentrationh precipitation on on theat the 20global global °C followedactivity activity recovery recoveryby 3 h of ofcrosslinking CGTase CGTase CLEAs CLEAs with (enzyme(enzymeglutaraldehyde solution solution under prepared prepared 200 rpm in in 50 stirring); 50 mM mM sodium sodium(B) influence citrat citratee ofbuffer buffercrosslinking pH pH 6.0, 6.0, timeprecipitation precipitation with 20 mM with with glutaraldehyde acetone acetone at ata ◦ volumetricaon volumetric the global concentration concentrationactivity recovery of of75%, 75%, (precipitation 1 1h hprecipitation precipitation and crosslinking at at20 20 °CC followed followedconditions by by at3 3 hthe h crosslinking crosslinking same conditions with with glutaraldehydeglutaraldehydeabove). under under 200 200 rpm rpm stirring); stirring); ( (BB)) influence inﬂuence of of crosslinking crosslinking time time with with 20 20 mM mM glutaraldehyde glutaraldehyde onon the globalglobal activityactivity recovery recovery (precipitation (precipitation and and crosslinking crosslinking conditions conditions at the at same the conditionssame conditions above). above). 100

100 75

75 50

50 25 Relative activity (%)

Relative activity (%) 0 0123 Time (h) 0 Figure 3. Effect of glutaraldehyde0123 modiﬁcation on the activity of free CGTase. Enzyme solution wasFigure prepared 3. Effect in of 50 glutaraldehyde mM sodium citratemodification buffer Timeon pH the 6.0 (h) activity in the of presence free CGTase. of 20 Enzyme mM glutaraldehyde. solution was Conditions:prepared in 50 enzyme mM sodium concentration citrate buffer of 0.355 pH 6.0 mg in protein/mL, the presence 20 of ◦20C mM at 200 glutaraldehyde. rpm stirring. Conditions: The initial Figureactivityenzyme 3. (11.71 concentrationEffect± of0.46 glutaraldehyde U/mL) of 0.355 was mg takenmodification protein/mL, as 100%. on 20 the°C atactivity 200 rpm of free stirring. CGTase. The Enzymeinitial activity solution (11.71 was ± prepared0.46 U/mL) in 50was mM taken sodium as 100%. citrate buffer pH 6.0 in the presence of 20 mM glutaraldehyde. Conditions: 2.4. Strategies to Improve the Global Activity Recovery of CGTase CLEAs enzyme concentration of 0.355 mg protein/mL, 20 °C at 200 rpm stirring. The initial activity (11.71 ± 2.4. Strategies to improve the global activity recovery of CGTase CLEAs 0.46Use U/mL) of feeder was taken proteins as 100%. during aggregation may reduce diffusion problems inside the CLEA structures and may facilitate the crosslinking of the enzyme molecules [23,44,46,47]. In addition, it was 2.4. Strategies to improve the global activity recovery of CGTase CLEAs

Catalysts 2018, 8, x FOR PEER REVIEW 5 of 17

Use of feeder proteins during aggregation may reduce diffusion problems inside the CLEA structures and may facilitate the crosslinking of the enzyme molecules [23,44,46,47]. In addition, it was previously demonstrated that cross-linking with macromolecular cross-linkers improves the mass transfer in the CLEAs [20,28,30]. According with the considerations mentioned above, in this work, BSA was evaluated as a protein feeder in the preparation of CLEAs of CGTase, using an enzyme/BSA mass ratio of 1:1. In addition, polyaldehydes (starch and pectin oxidized with periodate) were evaluated as macromolecular cross-linkers. It was observed that the addition of BSA in the preparation of CGTase CLEAs using glutaraldehyde as cross-linker (Figure 4) only slightly improved the global activity recoveryCatalysts 2019 after, 9, CLEA 120 preparation (global activity recovery only increased from 10% to 13%). 5 of 17 CLEAs prepared with aldehyde–starch and aldehyde–pectin as cross-linkers in the absence of BSA were approximately 3 and 2 times (respectively) more active than those prepared with previously demonstrated that cross-linking with macromolecular cross-linkers improves the mass glutaraldehyde. However, the crosslinking of the co-aggregates of CGTase and BSA with aldehyde– transfer in the CLEAs [20,28,30]. starch caused a decrease in CLEA activity (by around twofold) in opposition of the effect of BSA According with the considerations mentioned above, in this work, BSA was evaluated as using glutaraldehyde. This behavior suggested that an intense crosslinking is the main cause of the a protein feeder in the preparation of CLEAs of CGTase, using an enzyme/BSA mass ratio of low activity recovery, very likely by the decrease in the pore diameters of the CLEA structures, and 1:1. In addition, polyaldehydes (starch and pectin oxidized with periodate) were evaluated as thereby increasing intraparticle diffusional limitations. macromolecular cross-linkers. It was observed that the addition of BSA in the preparation of CGTase Thus, the highest global activity recovery (25.4 ± 1.4%) in the CLEAs of CGTase was obtained CLEAs using glutaraldehyde as cross-linker (Figure4) only slightly improved the global activity using aldehyde–starch as cross-linker. Consequently, aldehyde–starch was selected as the cross- recovery after CLEA preparation (global activity recovery only increased from 10% to 13%). linker for further investigation.

5 Global activity recovery (%)

0 e h A in SA rc t yd B a h t +BS ec s h -p e- rc d a yde aralde hy t t -s eh ld Glu Alde yde A Cross-linkers Glutaraldehyde+ Aldeh FigureFigure 4. 4. EffectEffect of of different different cross-linkers cross-linkers on on the the global global activity activity recovery recovery of of CLEAs CLEAs of of CGTase. CGTase. Reaction Reaction conditions:conditions: enzyme enzyme solution solution prepared prepared in in 50 50 mM mM sodium sodium citrate citrate buffer buffer pH pH 6.0, 6.0, precipitation precipitation with with ◦ acetoneacetone (75% (75% volumetric volumetric concentration, concentration, 20 20 °C,C, 1 1h) h) in in the the absence absence or or presence presence of of bovine bovine serum serum albumin albumin ◦ (BSA)(BSA) as as co-feeder co-feeder (enzyme/BSA (enzyme/BSA mass mass ratio ratio of of 1:1); 1:1); cr crosslinkingosslinking at at 20 20 °CC for for 3 3h hunder under 200 200 rpm rpm stirring. stirring.

CLEAs prepared with aldehyde–starch and aldehyde–pectin as cross-linkers in the absence of BSA The effect of the aldehyde–starch concentration on the CLEA activity was evaluated by varying thewere concentration approximately of the 3 and cross-linker 2 times (respectively) from 10 to 80 moremM. In active this thanset of those experiments prepared sodium with glutaraldehyde. borohydride wasHowever, added at the the crosslinking end point of of the the reaction co-aggregates to a fina ofl CGTaseconcentration and BSA of 1 with mg/mL, aldehyde–starch aiming to reduce caused the a Schiffdecrease bases in CLEAestablished activity between (by around aldehyde twofold) groups in opposition of the cross-linker of the effect ofand BSA amino using groups glutaraldehyde. of the proteinsThis behavior to secondary suggested amino that bonds, an intense and also crosslinking to reduce isthe the remnant main cause aldehydes of the of low the activity cross-linker recovery, to inertvery hydroxyl likely by groups the decrease [48]. in the pore diameters of the CLEA structures, and thereby increasing intraparticleAs shown diffusional in Figure 5A, limitations. the global activity recoveries of the CGTase CLEAs were very similar at ± the aldehyde–starchThus, the highest concentrations global activity of 10 recovery and 20 mM, (25.4 and1.4%) then instarted the CLEAs to decrease. of CGTase Reduced was CGTase obtained CLEAsusing aldehyde–starchwith the highest as cross-linker.activity (global Consequently, activity aldehyde–starchrecovery of 16.3%) was selectedwere obtained as the cross-linker using a concentrationfor further investigation. of 20 mM aldehyde–starch and 1 mg/mL of sodium borohydride in the cross-linking and reductionThe effect steps, of the respectively. aldehyde–starch concentration on the CLEA activity was evaluated by varying the concentration of the cross-linker from 10 to 80 mM. In this set of experiments sodium borohydride was added at the end point of the reaction to a ﬁnal concentration of 1 mg/mL, aiming to reduce the Schiff bases established between aldehyde groups of the cross-linker and amino groups of the proteins to secondary amino bonds, and also to reduce the remnant aldehydes of the cross-linker to inert hydroxyl groups [48]. As shown in Figure5A, the global activity recoveries of the CGTase CLEAs were very similar at the aldehyde–starch concentrations of 10 and 20 mM, and then started to decrease. Reduced CGTase CLEAs with the highest activity (global activity recovery of 16.3%) were obtained using a concentration of 20 mM aldehyde–starch and 1 mg/mL of sodium borohydride in the cross-linking and reduction steps, respectively. Catalysts 2018, 8, x FOR PEER REVIEW 6 of 17

We also evaluated the reduction with sodium borohydride in the presence of β-CD, maltose, and maltotriose, because these compounds (product or inhibitor of the CGTase) might protect the active site of the enzyme against deleterious effect of borohydride [49–51]. The presence of β-CD and maltotriose did not improve the global activity recoveries of the CGTase CLEAs in the reduction step with sodium borohydride. Either in absence and presence of the additives the global activity recovery was around 16%. However, the addition of maltose allowed a slight but significant increase of activity recovery (from 16 ± 0.4 % to 18 ± 0.6 %). Figure 5B shows that the cross-linking occurred rapidly, thus 2 h of crosslinking time were sufficient to obtain CLEAs with a global activity recovery around 20 ± 0.5%. After this time, the global activity recovery did undergo a small decrease, again suggesting that an intense crosslinking might Catalystsclose2019 the, pores9, 120 inside the CLEA, and this presented a negative effect on the observed enzyme activity.6 of 17

20 25 (A) (B)

20 15

15 10 10

5 5 Global activity recovery (%) recovery activity Global Global activity recovery (%) 0 0 10 20 40 80 2468 Cross-linking time (h) Aldehyde-starch concentration (mM) Figure 5. (A) Influence of aldehyde–starch concentration and (B) effect of the cross-linking time (20 mM aldehyde–starchFigure 5. (A) Influence in the presence of aldehyde–starc of 1 mg/mL maltose)h concentration on the global and (B) activity effect recovery of the cross-linking of CGTase CLEAs. time (20 ReactionmM aldehyde–starch conditions: enzyme in the solution presence prepared of 1 mg/mL in 50 maltose) mM sodium on the citrate global buffer activity pH recovery 6.0, precipitation of CGTase ◦ ◦ withCLEAs. acetone Reaction (75% volumetric conditions: concentration, enzyme solution 20 C, prepared 1 h); crosslinking in 50 mM at 20sodiumC for citrate 3 h under buffer 200 pH rpm 6.0, ◦ stirring;precipitation reduction with with acetone sodium (75% borohydride volumetric (1 concentrat mg/mL, 30ion, min, 204 °C,C). 1 h); crosslinking at 20 °C for 3 h under 200 rpm stirring; reduction with sodium borohydride (1 mg/mL, 30 min, 4 °C). We also evaluated the reduction with sodium borohydride in the presence of β-CD, maltose, and maltotriose, because these compounds (product or inhibitor of the CGTase) might protect the Although the reduction of CLEAs with sodium borohydride produces secondary amino bonds active site of the enzyme against deleterious effect of borohydride [49–51]. between aldehyde–starch and protein, the incubation of the CLEAs in 50 mM citrate buffer (pH 6.0) The presence of β-CD and maltotriose did not improve the global activity recoveries of the CGTase for 1 h led to a partial leaching of the enzyme (∼30%). This drawback was overcome by adding CLEAs in the reduction step with sodium borohydride. Either in absence and presence of the additives polyethylenimine at the end point of the cross-linking step [22]. This treatment only increased the the global activity recovery was around 16%. However, the addition of maltose allowed a slight but global activity recovery from 18 ± 0.6 to 24 ± 0.4 %, but it allowed the formation of more robust CLEAs, significant increase of activity recovery (from 16 ± 0.4 % to 18 ± 0.6 %). fully preventing the enzyme leakage. FigureIn summary,5B shows the that most the cross-linkingactive CLEA occurredof CGTAse rapidly, (around thus 24% 2 h global of crosslinking activity recovery) time were was ± sufficientprepared to obtainas follows: CLEAs enzyme with a globalsolution activity prepared recovery in 50 around mM sodium 20 0.5%. citrate After buffer this time, pH the6.0, global protein activityprecipitation recovery with did acetone undergo (75% a small volumetric decrease, concentr again suggestingation, 20 °C, that 1 anh); intensecrosslinking crosslinking with aldehyde– might closestarch the pores(20 mM, inside 20 °C, the 2 CLEA, h); subsequent and this presented addition aof negative polyethylenimine effect on the (1 observed mg/mL, enzyme20 °C, 5 activity.min) and reductionAlthough with the sodium reduction borohydride of CLEAs with(1 mg/mL, sodium pH borohydride 6.0, 4 °C, 30 produces min, in secondarythe presence amino of 1 bonds mg/mL betweenmaltose). aldehyde–starch All steps were andperformed protein, under the incubation 200 rpm shaking. of the CLEAs in 50 mM citrate buffer (pH 6.0) for 1 hIt led has to been a partial reported leaching low activity of the enzymerecoveries (~30%). (<32%) Thisfor covalent drawback immobilization was overcome of CGTase by adding from polyethylenimineThermoanaerobacter at thesp. endon pointEupergit of the C cross-linking(10.2%) [34], stepglyoxyl-activated [22]. This treatment silica (1.5%) only increased [36], glyoxyl- the ± ± globalactivated activity agarose recovery (32%) from [37], 18glutaraldehyde-activated0.6 to 24 0.4 %, but chitosan it allowed (6.1%) the [38], formation glutaraldehyde-activated of more robust CLEAs,silica fully(< 6%), preventing CNBr-activated the enzyme Sepharose leakage. (4.4%), and 1,6-diaminohexane-activated Sepharose (2.4%) [41].In The summary, main causes the most for active the low CLEA recovered of CGTAse activiti (aroundes commonly 24% global reported activity for recovery) covalent wasimmobilization prepared as follows: enzyme solution prepared in 50 mM sodium citrate buffer pH 6.0, protein precipitation of CGTase are probably as follows: (i) intraparticle◦ diffusional limitations due to the large molecular withsize acetone of substrate (75% (starch) volumetric and concentration,products (cyclodextrins 20 C, 1 h);); (ii) crosslinking complexity with of the aldehyde–starch CGTase 3D-structure (20 mM, and 20 ◦C, 2 h); subsequent addition of polyethylenimine (1 mg/mL, 20 ◦C, 5 min) and reduction with its catalytic mechanism, because this enzyme◦ has five domains (A to E) and catalyze four possible sodiumreactions borohydride (hydrolysis, (1 mg/mL, cyclization, pH 6.0,coupling, 4 C, 30 and min, di insproportionation) the presence of 1[5,52]; mg/mL (iii) maltose). the modification All steps of werecertain performed Lys residues under 200involved rpm shaking. in the catalytic mechanism of the CGTase [41]. Martin et al. [41] reportedIt has been that reportedsome of lowthe Lys activity residues recoveries in the (<32%) starch-binding for covalent site immobilization(Lys547 and Lys549 of) CGTase display froma high Thermoanaerobacter sp. on Eupergit C (10.2%) [34], glyoxyl-activated silica (1.5%) [36], glyoxyl-activated agarose (32%) [37], glutaraldehyde-activated chitosan (6.1%) [38], glutaraldehyde-activated silica (<6%), CNBr-activated Sepharose (4.4%), and 1,6-diaminohexane-activated Sepharose (2.4%) [41]. The main causes for the low recovered activities commonly reported for covalent immobilization of CGTase are probably as follows: (i) intraparticle diffusional limitations due to the large molecular size of substrate (starch) and products (cyclodextrins); (ii) complexity of the CGTase 3D-structure and its catalytic mechanism, because this enzyme has five domains (A to E) and catalyze four possible reactions (hydrolysis, cyclization, coupling, and disproportionation) [5,52]; (iii) the modification of certain Lys residues involved in the catalytic mechanism of the CGTase [41]. Martin et al. [41] reported that some of the Lys residues in the starch-binding site (Lys547 and Lys549) display a Catalysts 2019, 9, 120 7 of 17 Catalysts 2018, 8, x FOR PEER REVIEW 7 of 17 accessibilityhigh accessibility to the tosolvent, the solvent, and are and thus are possible thus possible targets for targets enzyme for immobilization. enzyme immobilization. Also, Wind Also, et 47 al.Wind [52] et reported al. [52] reported an important an important stabilizer stabilizer role of role Lys of47 Lys in thein theactive active site site of ofthe the CGTase CGTase from from ThermoanaerobacteriumThermoanaerobacterium thermosulfurigenesthermosulfurigenesEM1, EM1, which which shares shares an overallan overall identity identity of 89% of with 89% the with CGTase the CGTasefrom Thermoanaerobacter from Thermoanaerobactersp. ATCC sp. 53627 ATCC [5]. 53627 Thus, [5]. we Thus, might we suppose might that suppose the involvement that the involvement of some of ofthese some Lys of residues these Lys in theresidues covalent in linkagethe covalent with thelinkage cross-linker with the (or cross-linker aldehyde-activated (or aldehyde-activated supports) could supports)drastically could reduce drastically the catalytic reduce efficiency the catalytic of the efficiency CGTase. of the CGTase. Surprisingly,Surprisingly, Matte Matte et et al. [33] [33] reported an acti activityvity recovery of 73% for covalent immobilization ofof CGTase CGTase from Thermoanaerobacter sp. on glutaraldehyde-activated silica. silica. In In this this case, case, mesoporous silicasilica microspheres microspheres were were synthesize synthesizedd using using polyethylene polyethylene glycol glycol 400 400 as as swelling swelling agent, agent, silanized silanized with with 3-aminopropyltrimethoxysilane3-aminopropyltrimethoxysilane (APTMS),(APTMS), and activatedand activated with glutaraldehyde with glutaraldehyde prior to immobilization. prior to immobilization.Although in our Although work the activityin our work recovery the activity was smaller, recovery the methodologywas smaller, the adopted methodology to immobilize adopted the toCGTase immobilize does notthe useCGTase solid does support not anduse solid thus, support saving on and the thus, cost saving of the supporton the cost may of reduce the support the impact may reduceof thebiocatalyst the impact productionof the biocatalyst costs in pr theoduction overall costs process. in the overall process.

2.5.2.5. Thermal Thermal inactivation Inactivation assay Assay TheThe thermal stabilitiesstabilities ofof free free CGTase CGTase and and the the most mo activest active CLEA CLEA of CGTase of CGTase prepared prepared in this in paper this ◦ paperwas evaluated was evaluated at 60 C andat 60 pH °C 6.0 and (50 mMpH sodium6.0 (50 citratemM sodium buffer). Althoughcitrate buffer). a high thermostabilityAlthough a high for ◦ thermostabilityThermoanaerobacter for Thermoanaerobactersp. CGTase at temperatures sp. CGTase below at temperatures 75 C[3] has below been 75 reported, °C [3] has some been inactivation reported, ◦ somewas observed inactivation for freewas CGTase observed at 60for Cfree (activity CGTase loss at of60around °C (activity 20% afterloss 6of h around as shown 20% in after Figure 6 6h) inas shownabsence in of Figure substrate. 6) in However, absence of no substrate. signiﬁcant Howeve loss of activityr, no significant was observed loss of to activity the CGTAse was CLEAobserved under to thethe CGTAse same inactivation CLEA under conditions. the same That inactivation way, the conditions. immobilized That enzyme way, the was immobilized signiﬁcantly enzyme more stable was significantlythan the free more enzyme. stable than the free enzyme.

100

20 Free CGTase Relative activity (%) CGTase CLEA

0 0123456 Time (h)

Figure 6. Thermal inactivation of free and immobilized CGTase at 60 ◦C and pH 6.0 (50 mM Figure 6. Thermal inactivation of free and immobilized CGTase at 60 °C and pH 6.0 (50 mM citrate citrate buffer). The initial activities of the enzyme solution or suspension (4.72 ± 0.05 U/mL and buffer). The initial activities of the enzyme solution or suspension (4.72 ± 0.05 U/mL and 1.89 ± 0.05 1.89 ± 0.05 U/mL, respectively) were taken as 100%. U/mL, respectively) were taken as 100%. 2.6. Cyclodextrin Production 2.6. Cyclodextrin production The most active CLEA of CGTase prepared in this paper was used for cyclodextrin production in a stirredThe most tank-type active bench CLEA reactor of CGTase equipped prepared with in an this impeller paper withoutwas used blades for cyclodextrin (Figure7). This production reactor inoperating a stirred astank-type a vortex bench ﬂow reactor reactor equipped may avoid with damage an impeller to shear without sensitive blades catalysts (Figure like 7). This CLEAs reactor [23]. operatingThe temperature as a vortex was flow set at reactor 50 ◦C may to avoid avoid thermal damage inactivation to shear sensitive of the free catalysts CGTase. like CLEAs [23]. The temperature was set at 50 °C to avoid thermal inactivation of the free CGTase.

Catalysts 2019, 9, 120 8 of 17 Catalysts 2018, 8, x FOR PEER REVIEW 8 of 17

Catalysts 2018, 8, x FOR PEER REVIEW 8 of 17

Figure 7. BenchBench reactor reactor equipped equipped with with an an impeller impeller with without out blades blades used in the production of cyclodextrinsFigure 7. Bench from reactor pre-hydrolyzed equipped with cassavacassava an starchstimpellerarch usingusing with CGTaseCGTaseout blades CLEACLEA used asas biocatalyst.biocatalyst.in the production of cyclodextrins from pre-hydrolyzed cassava starch using CGTase CLEA as biocatalyst. ◦ Figure8 8 shows shows the the reaction reaction course course in in the the production production of of CDs CDs ( α(α-,-,β β-,-, and andγ γ-CD)-CD) atat 5050°C and pH 6.0, catalyzedFigure 8 shows by CGTase the reaction (free oror course inin thethe in formform the production of CLEAs) usingof CDs 2.0% (α-, β ((w/v)w-,/ andv) pre-hydrolyzed γ-CD) at 50°C and starch pH (DE 1.4%).6.0, catalyzed It can be by observed CGTase (free that or CGTase in the form CLEAsCLEAs of CLEAs) producedproduced using similar 2.0% amounts(w/v) pre-hydrolyzed of CDs, but starch the maximum (DE concentrations1.4%). It can be ofobservedof total total CDs CDs that (around CGTase(around 9.0 CLEAs g/L)9.0 g/L) were pr oducedwere reached reached similar at different amountsat different reaction of CDs, reaction times. but the Aftertimes. maximum 6 hAfter reaction, 6 h usingreaction,concentrations free using CGTase, offree total theCGTase, concentrationsCDs (aroundthe concentrations 9.0 of g/L)α-, β were-, andof αreached-,γ -CDβ-, and reached at γ -CDdifferent 3.3,reached 3.8,reaction and 3.3, 1.9 3.8,times. g/L and After (1.9α/ βg/L /6γ h(massα/β/γ ratiomassreaction, ofratio 1.7:2:1), using of 1.7:2:1), free which CGTase, which correspond the correspond concentrations to a to starch-to-CDs a starch-to-CDs of α-, β-, and yield γ yield-CD of reached 45%.of 45%. On 3.3, On the 3.8, the otherand other 1.9 hand, hand, g/L (α/β/γ afterafter 33 h reaction,mass ratio using using of 1.7:2:1), CLEAs CLEAs which of of CGTase, CGTase,correspond the the toCD CD a starch-to-CDsco concentrationsncentrations yield were were of 2.7,45%. 2.7, 4.0 On 4.0 and the and 1.8other 1.8 g/L g/Lhand, (α/β/γ (α after/ massβ/ 3γ h massratio α/β/γ ratioofreaction, 1.5:2:1), of 1.5:2:1), using which CLEAs which correspond of correspond CGTase, to athe tostarch-to-CDs CD a starch-to-CDs concentrations yield yield wereof 43%. of2.7, 43%. Similar4.0 and Similar 1.8findings g/L ﬁndings ( were mass were reported ratio reported by byMartínof Mart1.5:2:1), etín al. etwhich [34] al. [ 34with correspond] with CGTase CGTase to from a fromstarch-to-CDs ThermoanaerobacterThermoanaerobacter yield of sp 43%.. immobilizedsp. immobilizedSimilar findings on Eupergit on were Eupergit C.reported However, C. However, by the themaximumMartín maximum et al. conversion [34] conversion with CGTase of 10% of 10%from(w/v) (Thermoanaerobacterw soluble/v) soluble potato potato st archsp. starchimmobilized into cyclodextrins into cyclodextrins on Eupergit (44%) C. (44%) wasHowever, reached was reachedthe after ◦ after48maximum h reaction 48 h reaction conversion at 60 at°C 60 andof 10%C pH and (w/v) 5.5 pH (10 5.5soluble mM (10 mMsodiumpotato sodium st citratearch citrate into buffer), cyclodextrins buffer), yielding yielding (44%) an α/β/γ an wasα /mass βreached/γ ratiomass after of ratio 3:5:1. of 48 h reaction at 60 °C and pH 5.5 (10 mM sodium citrate buffer), yielding an α/β/γ mass ratio of 3:5:1. 3:5:1.The immobilization The immobilization may alter may alterthe catalytic the catalytic features features of an of enzyme, an enzyme, by some by some enzyme enzyme distortion distortion or by or The immobilization may alter the catalytic features of an enzyme, by some enzyme distortion or by bythe thegeneration generation of ofdiffusional diffusional limitations limitations [11]. [11 ].CGTase CGTase has has a avery very complex complex catalytic catalytic mechanism mechanism and the generation of diffusional limitations [11]. CGTase has a very complex catalytic mechanism and tridimensional structure [[5,53]5,53] thatthat couldcould bebe changedchanged and/orand/or sterically hindered depending on the tridimensional structure [5,53] that could be changed and/or sterically hindered depending on the immobilization type andand therebythereby alteringaltering itsits normalnormal catalyticcatalytic action.action. immobilization type and thereby altering its normal catalytic action.

10 (A) Total yield ∼ 43% 4.0 (B) 10 (A) Total yield ∼ 43% 4.0 (B) 3.5 3.5 8 3.0 3.0 2.5 6 2.5 2.0 2.0 4 4 1.5 1.5

1.0 1.0 2 Free Free CGTase CGTase Free CGTase Free CGTase CGTase CGTase CLEA CLEA 0.5 0.5 CGTase CGTase CLEA CLEA

0 0.0

0 (g/L) concentration -Cyclodextrin 0.0 -Cyclodextrin concentration (g/L) concentration -Cyclodextrin Total cyclodextrin concentration (g/L) concentration cyclodextrin Total

0123456 α 0123456 Total cyclodextrin concentration (g/L) concentration cyclodextrin Total

0123456 α 0123456 Time (h) Time (h) TimeTime (h) (h) Figure 8. Cont.

CatalystsCatalysts2019 2018, 9, ,8 120, x FOR PEER REVIEW 9 of 179 of 17

4.5 (C) 2.1 (B) 4.0

3.5 1.8

3.0 1.5

2.5 1.2 2.0 0.9 1.5 0.6 1.0 Free CGTase Free CGTase 0.3 CGTase CLEA 0.5 CGTase CLEA

-Cyclodextrin concentration (g/L) concentration -Cyclodextrin 0.0 β

0.0 (g/L) concentration -Cyclodextrin

γ 0123456 0123456 Time (h) Time (h)

FigureFigure 8. 8.Batch Batch assaysassays ofof (A) total CDs, CDs, ( (BB)) αα-CD,-CD, (C (C) )ββ-CD,-CD, and and (D (D) γ)-CDγ-CD production production at 50 at ° 50C and◦C andpH pH 6.06.0 for for 6 6 h, h, catalyzed catalyzed byby free CGTase (5.0 U/g U/g starch starch)) and and CLEAs CLEAs of ofCGTase CGTase (5.0 (5.0 U/g U/g starch). starch). Substrate: Substrate: 2.0%2.0% ( w(w/v)/v) pre-hydrolyzed pre-hydrolyzed starch starch (DE (DE 1.4%). 1.4%). One On Unite Unit (U) correspond(U) correspond the activity the activity of CGTase of CGTase measured withmeasured 0.5% ( wwith/v) 0.5% dextrin (w/v) as dextrin substrate, as su asbstrate, described as described in Section in 3.2 Section. 3.2.

TheThe operational operational stability stability ofof CLEAsCLEAs ofof CGTaseCGTase waswas evaluatedevaluated in fivefive reaction cycles of 3 3 h at at 50 50 ◦C and°C and pH 6.0pH (Figure6.0 (Figure9). 9). The The total total CD CD yield yield decreased decreased fromfrom 42.6% (first (first cycle) cycle) to to 34.6% 34.6% (fifth (fifth cycle), cycle), aa decrease decrease of of 18.8% 18.8% inin thethe catalytic efficiency efficiency of of th thee enzyme. enzyme. This This decrease decrease did did not not affect affect similarly similarly to to β α γ allall CDs. CDs. The The yields yields ofof β-CD decreased to to 66%, 66%, of of the the initial initial ones, ones, while while for for -α and- and -CDγ-CD the thedecrease decrease β waswas to to 94% 94% of of the the initialinitial productions.productions. The The drop drop in in the the β-CD-CD yield yield was was much much more more pronounced pronounced in the in the secondsecond cycle cycle (a (a decreasedecrease ofof 23%).23%). This This suggest suggest th thee complex complex catalytic catalytic mechanism mechanism of this of this enzyme enzyme and and revels that an additional problem is that together to the reaction rate, the quality of the product may revels that an additional problem is that together to the reaction rate, the quality of the product may be different in each reaction cycle. In fact, partial and controlled inactivation of the enzyme under be different in each reaction cycle. In fact, partial and controlled inactivation of the enzyme under different conditions may be an interesting way to modulate the enzyme selectivity for the different different conditions may be an interesting way to modulate the enzyme selectivity for the different CDs, as each inactivation condition may produce different enzyme changes [54]. CDs, as each inactivation condition may produce different enzyme changes [54]. The operational stability of CGTase from Thermoanaerobacter sp. immobilized on activated supportsThe operational was previously stability studied. of CGTase Martín from et al.Thermoanaerobacter [34] reported a losssp. of immobilized60% in the catalytic on activated efficiency supports of wasthepreviously enzyme immobilized studied. Mart on íEupergitn et al. [34 C] after reported 10 cycles a loss of of 24 60% h at in 60 the °C catalytic and pH efficiency5.5 (around of 50% the enzymeloss ◦ immobilizedafter five cycles). on Eupergit Schoffer Cet afteral. [38] 10 reported cycles ofa loss 24 h of at 40% 60 inC the and production pH 5.5 (around of β-CD 50% from loss 4% after(w/v) five cycles).soluble Schofferstarch after et al.100 [38 cycles] reported of 15 min a loss at 60 of 40%°C and in pH the 6.0 production (around 10% of β lo-CDss after from 5 4%cycles) (w/ forv) soluble the ◦ starchCGTase after immobilized 100 cycles on of glutaraldehyde-activated 15 min at 60 C and pH 6.0chitosan. (around Matte 10% et lossal. [33] after reported 5 cycles) a loss for of the 40% CGTase in immobilizedthe production on of glutaraldehyde-activated β-CD from 4% (w/v) starch chitosan. after 15 cycles Matte of et 15 al. min [33 ]at reported 80 °C and a losspH 6.0 of (around 40% in the production5% loss after of β five-CD cycles) from 4% for (w the/v )enzyme starch after immobilized 15 cycles ofon 15glutaraldehyde-activated min at 80 ◦C and pH 6.0 silica. (around The 5% lossoperational after five stability cycles) forof CLEAs the enzyme of CGTase immobilized (around on20% glutaraldehyde-activated loss after five cycles of 3 silica.h at 50 The °C operationaland pH stability6.0) was of good CLEAs compared of CGTase to the (around ones with 20% CGTase loss after immobilized five cycles on of solid 3 h atsupports. 50 ◦C and pH 6.0) was good compared to the ones with CGTase immobilized on solid supports.

Catalysts 2018, 8, x FOR PEER REVIEW 10 of 17 Catalysts 2019, 9, 120 10 of 17

10 α:β:γ = 1.5:2.1:1 Total CDs α:β:γ α:β:γ = 1.5:1.5:1 α YCD = 42.6% = 1.6:1.7:1 -CD Y = 35.9% YCD = 38.1% α:β:γ = 1.5:1.5:1 CD α:β:γ = 1:5:1.5:1 β-CD Y = 34.6% γ 8 YCD = 35.1% CD -CD

2 Concentration (g/L)

0 12345 6Cycle h-Cycle number

2 Concentration (g/L) Concentration

0 12345 6 h-Cycle Cycle number

Figure 9. Reusability of CGTase CLEAs in cyclodextrin production at 50 ◦C and pH 6.0, using an enzyme load of 5.0 U/g starch and 2.0% (w/v) pre-hydrolyzed starch (DE = 1.43) as substrate. The notes at the top of the columns represent the mass ratios of CDs (α/β/γ) produced and the starch-to-CD yieldsFigure in 9. each Reusability batch. Each of CGTase cycle takes CLEAs 3 h. in cyclodextrin production at 50°C and pH 6.0, using an enzyme load of 5.0 U/g starch and 2.0% (w/v) pre-hydrolyzed starch (DE = 1.43) as substrate. The 3. Materialsnotes at andthe top Methods of the columns represent the mass ratios of CDs (α/β/γ) produced and the starch-to- CD yields in each batch. Each cycle takes 3 h. 3.1. Materials 3. MaterialsThermostable and Methods CGTase from Thermoanaerobacter sp. (Toruzyme®3.0L, Novozymes, Denmark), containing 7.1 mg protein/mL with a speciﬁc activity (initial rate of β-cyclodextrin production) of3.1. around Materials 30 U/mg protein and α-amylase from Bacillus amyloliquefaciens (BAN®480L) were from NovozymesThermostable (Bagsvaerd, CGTase Denmark). from Thermoanaerobacterα-, β-, and γ-Cyclodextrin, sp. (Toruzyme® dextrin 3.0L, 10, Novozymes, bovine serum Denmark), albumin (BSA)containing and polyethylenimine7.1 mg protein/mL (oligomer with a specific mixture, activity Mn ~423)(initial were rate purchasedof β-cyclodextrin from Sigma-Aldrichproduction) of (St.around Louis, 30 MO, U/mg USA). protein Soluble and starch, α-amylase phenolphthalein, from Bacillus and amyloliquefaciens glutaraldehyde (BAN® (25% v/ 480L)v) were were obtained from fromNovozymes Synth (Diadema, (Bagsvaerd, SP, Brazil).Denmark). The α other-, β-, reagentsand γ-Cyclodextrin, were of analytical dextrin grade. 10, bovine serum albumin (BSA)Protein and polyethylenimine concentration was (oligo determinedmer mixture, by Bradford’s Mn ~423) method were purc [55]hased using from bovine Sigma-Aldrich serum albumin (St. (BSA)Louis, as MO, standard USA). protein.Soluble starch, phenolphthalein, and glutaraldehyde (25% v/v) were obtained from Synth (Diadema, SP, Brazil). The other reagents were of analytical grade. 3.2. Enzymatic Activity Assay Protein concentration was determined by Bradford’s method [55] using bovine serum albumin (BSA)Enzymatic as standard activity protein. of soluble and immobilized CGTase was determined by measuring the initial reaction rate of β-CD formation using dextrin as substrate. A volume of 55 mL dextrin 10 (0.5% w/v) in3.2. 10 Enzymatic mM citrate activity buffer assay (pH 6.0) was added to a batch reactor that was thermostatized at 60 ◦C, because

Catalysts 2019, 9, 120 11 of 17 the high thermostability of Thermoanaerobacter sp. CGTase at temperature ≤ 75 ◦C[3]. One hundred microliters of 50x diluted enzyme solution (soluble) or 300 µL of immobilized enzyme suspension were added to the reaction medium and aliquots of 1 mL were withdrawn at 2-min intervals for 10 min. The enzymatic reaction was stopped by adding 20 µL of 3 M HCl followed by 5 min of boiling. β-CD concentration was determined using the colorimetric method of phenolphthalein, developed in 1981 by Vikmon [56] and modiﬁed as described by Tardioli et al. [37]. The assays were carried out by mixing 200 µL of the sample containing β-CD and 1.0 mL of a solution of 0.06 M phenolphthalein, prepared in 120 mM sodium bicarbonate buffer (pH 10.5). This solution was prepared mixing 0.3 mL of phenolphthalein solution (3 mM in 95% ethanol) and 14.7 mL of 120 mM sodium bicarbonate buffer, pH 10.5. The absorbance of the samples was record at 550 nm (Spectrophotometer UV-Visible Ultrospec 200, Pharmacia Biotech, Piscataway, NJ, USA). A blank was used, substituting the sample by distilled water. The β-CD concentration (Cβ-CD, in mM) was determined from a calibration curve of β-CD ranging from 0 to 1 mM. Equation (1) was ﬁtted to the absorbance data as a function of the β-CD concentrations using the software Origin 8.0: A550 A0/550 Cβ−CD = 6000 a 1 − 1 + , (1) A0/550 Kβ−CDaA550

−5 where a is the total concentration of phenolphthalein in the assay cuvette 5 × 10 M ;A550 and −1 A0/550 are the absorbance of samples and blank, respectively; and Kβ−CD (16193.82 ± 112.95 M ) is the equilibrium constant determined by ﬁtting Equation (1) to the experimental data, and 6000 is the sample dilution from assay tube to the cuvette multiplied by the molar to milimolar conversion factor. One unit (U) was deﬁned as the amount of enzyme that produces 1 µmol of β-CD per minute under the assay conditions.

3.3. CLEAs Preparation

3.3.1. Precipitant Selection A volume of 900 µL of the precipitating agent (ethanol ≥ 99.5%, isopropanol ≥ 99.7%, methanol ≥ 99.9%, tert-butanol ≥ 99.0%, acetonitrile ≥ 99.8%, acetone ≥ 99.9%, or saturated ammonium sulfate solution) was added to 100 µL enzyme solution (3.55 mg protein/mL in 50 mM sodium citrate buffer pH 6.0) into a 2 mL micro-tube to a ﬁnal concentration of 90% (v/v). After incubation at 20 ◦C and 150 rpm stirring for 1 h, the suspension was centrifuged at 4 ◦C and 900 rpm for 10 min. The precipitate was re-dissolved in 1 mL of 50 mM sodium citrate buffer (pH 6.0) and its enzymatic activity and protein concentration were determined using colorimetric phenolphthalein and Bradford’s methods, respectively. The precipitant was chosen based on its precipitation yield in terms of activity (YPA), calculated as Equation (2): total redissolved activity YP = × 100, (2) A total initial activity For the selected precipitant (acetone in this case) it was also evaluated its concentration ranging from 75% to 90% (v/v).

3.3.2. Cross-Linking Using Glutaraldehyde After enzyme aggregation step, glutaraldehyde (25%, v/v) was added to the mixture to ﬁnal concentrations of 10, 20, 30, or 50 mM. The stirring at 200 rpm was maintained for 3 h at 20 ◦C. The mixture was centrifuged at 4000 rpm and the supernatant was separated from the CLEAs. The precipitate was re-suspended in 50 mM sodium citrate buffer pH 6.0 and centrifuged again. This procedure was repeated until disappearance of enzymatic activity in the supernatant. Catalysts 2019, 9, 120 12 of 17

The effect of the reaction time in the reaction of the enzyme aggregates with glutaraldehyde was studied by ranging the incubation period of the CLEAs at 20 ◦C from 1 to 4 h and maintaining the concentration of glutaraldehyde previously selected. The CLEA activity was measured using the colorimetric method of phenolphthalein, as described above. The global activity recovery (ARG) of all CGTase CLEAs was calculated as Equation (3):

Total activity of the CLEA AR = × 100, (3) G Total initial activity

3.4. Evaluation of Macromolecular Cross-Linkers

3.4.1. Preparation of Macromolecular Cross-Linkers Starch–aldehyde and pectin–aldehyde were prepared following the methodology described by Zhen et al. [30] with some modiﬁcations. A mass of 1.5 g of starch was solubilized in 50 mL of distilled water by heating for 3 min in a boiling water bath. Afterwards, the solution was cooled (until 30 ◦C), the pH was adjusted to 4.0, and 3.85 g of sodium periodate was added for complete oxidation of diol groups to aldehyde (120 min reaction). After, the solution was transferred to a 1 kDa cut-off membrane dialysis and dialyzed against distilled water at room temperature for 24 h under stirring and with six changes of distilled water. Under these conditions, the starch–aldehyde solution had a concentration of 320 mM of aldehyde groups, determined by sodium periodate consumption. For the preparation of pectin–aldehyde, the pectin (1.5 g) was dissolved in 20% (v/v) ethanol solution, and the other steps were identical as described above.

3.4.2. Cross-Linking Procedure Using Macromolecular Cross-Linkers The cross-linking with macromolecular cross-linkers (starch–aldehyde and pectin–aldehyde) was carried out at 20 ◦C by adding an amount of macromolecular cross-likers to the mixture of precipitant and enzyme (75% precipitant volumetric concentration) to a final concentration of 20 mM of aldehyde groups. The CLEA suspension was stirred at 200 rpm for 3 h. After, the CLEAs were recovered by centrifugation and washed with 50 mM citrate buffer (pH 6.0) as described above. The effect of the concentration of starch–aldehyde on the properties of the final CLEA was studied by ranging its concentration from 10 to 80 mM of aldehyde groups, followed by reduction with sodium borohydride (1 mg/mL) at 4◦C and pH 6.0 for 30 min. For the best starch–aldehyde concentration, the reduction step in the presence of additives (β-CD, maltose, and maltotriose) at 1 mg/mL was evaluated. The effect of the reaction time with aldehyde–starch was also evaluated ranging the reaction time from 2 to 8 h maintaining the other parameters previously selected. For the best selected condition, it was evaluated the treatment of the CLEAs with polyethylenimine (PEI). In this case, PEI was added to the CLEA preparation medium to a final concentration of 1 mg/mL after the precipitating and crosslinking steps. After 5 min reaction, the reduction step with sodium borohydride follows as described above.

3.5. Production of Cyclodextrins CDs were produced from pre-hydrolyzed starch (2.0% w/v, Dextrose Equivalent of 1.4%) at 50◦C in a batch reactor stirred with an impeller without blades, operating similarly as a batch Vortex Flow Reactor (VFR) in turbulent regime [57–59] with a cylinder rotation rate (ω) of 900 rpm. This reactor was dimensioned with a radius ratio (η = Ri/Re) of 0.24 and aspect ratio (Γ = L/d) of 6.32 [23]. Initially, a solution of starch (2.0%, w/v, 500 mL), prepared in 50 mM sodium citrate buffer pH 6.0 was gelatinized at 85◦C for 10 min. After, the temperature was adjusted to 60◦C and 3 µL of α-amylase (BAN®480L, Novozymes Company, Bagsvaerd, Denmark) were added (0.3 mL of α-amylase/kg of starch. After 5 min reaction, the pH was adjusted to 3.0 using 5 M HCl to inactivate the enzyme, and ﬁnally adjusted to 6.0 [60]. The total amount of reducing sugar (TRS) was determinate Catalysts 2019, 9, 120 13 of 17 by DNS method [61] using glucose as standard, and the dextrose equivalent (DE) was calculated using Equation (4): mass of TRS DE = × 100, (4) mass of starch The reactions of CD production were carried out for 6 hours at pH 6.0 (50 mM sodium citrate buffer), using an enzyme loading of 5.0 U/g of pre-hydrolyzed starch (30 mL starch solution). The temperature, enzyme loading, and reaction time for the production of CDs were selected based on previous studies with soluble enzyme [60]. Samples were withdrawn at 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 h for analysis of the α-, β- and γ-CD concentrations by ELSD-HPLC chromatographic method [62]. The total yield of CD production (YCD) was calculated following Equation (5):

g CD concentration in L YCD = g × 100, (5) Starch concentration in L

3.6. Thermal and Operational Stability The thermal stability of free and immobilized CGTase was evaluated at 60 ◦C and pH 6.0 (50 mM sodium citrate buffer) in the absence of substrate. A solution of free CGTase (4.72 ± 0.05 U/mL) or a suspension of CLEAs of CGTase (1.89 ± 0.10 U/mL) was incubated at the temperature above and samples were taken at 2 h intervals within 6 h for activity measurement as described in Section 3.2. The operational stability of the CLEAs of CGTase was evaluated using a pre-hydrolyzed starch solution (2.0% w/v, Dextrose Equivalent of 1.4%) in 50 mM citrate buffer pH 6.0. The reactions were performed for 3 h at 50 ◦C using an enzyme loading of 5.0 U/g pre-hydrolyzed starch (30 mL starch solution). At the end of each batch (ﬁve 3-h cycles), the biocatalyst was separated from the reaction medium by centrifugation and washed with distilled water. The CDs were analyzed by ELSD-HPLC method [62].

3.7. Chromatographic Method for Analysis of CDs The concentrations of α-, β-, and γ-CDs were determined using the chromatographic method described by Rojas et al [62]. Brieﬂy, the CDs were separated through a pyramid NUCLEODUR® C18 column (150 mm × 4.6 mm, 5 µm, Macherey-Nagel, Düren, Germany), at 30 ◦C, using acetonitrile (solvent B) and water, containing 1% (v/v) acetic acid (solvent A) at a ﬂow rate of 0.3 mL/min, using a linear gradient, as follows: 0–20 min with 100 to 80% of A and 0 to 20% of B; 20.1–30 min with 100% of A. All analyzes were performed using an Alliance E2695 liquid chromatograph (Waters, MI, USA) equipped with an evaporative light scattering detector (ELSD 2424, Waters, MI, USA).

4. Conclusions The results presented in this work showed that the free-carrier aggregation/crosslinking is a promising method for immobilizing CGTase when properly performed. It was demonstrated that the type of precipitant, precipitant concentration, cross-linking time, type and concentration of cross-linker greatly influenced the final activity of the CGTase CLEAs. Compared with the traditionally used glutaraldehyde, the preparation of CLEAs using alternative macromolecular cross-linkers allowed increasing the activity recovery. Addition of polyethylenimine after cross-linking step allowed avoiding the lixiviation of enzyme to reaction medium. The application of CGTase CLEAs in cyclization reaction from pre-hydrolyzed starch allowed a yield of CDs around 45%. CLEAs of CGTase might be easily recovered from the reaction medium by gravitational sedimentation or centrifugation and re-used in five 3-h cycles of cyclization (at 50 ◦C and pH 6.0), retaining around 80% of the initial productions of CDs. The use of a batch reactor operating similarly as a batch vortex flow reactor in turbulent regime avoided loss of biocatalyst by shear, a problem in conventional stirred tank reactors using shear sensitive particles. The system presented in this work, using free-carrier CGTase and vortex flow reactor, may be promising in the production of CDs as alternative to the systems using CGTase Catalysts 2019, 9, 120 14 of 17 immobilized on shear resistant particles to be used in stirred tank reactors, because saving on the cost of the support may reduce the impact of the biocatalyst production costs in the overall process.

Author Contributions: M.J.R. performed all experimental assays; M.A.-F. helped in the inactivation and reuse assays; R.d.L.C.G., G.M.Z., R.F.-L., and P.W.T. designed and supervised all experiments, as well as wrote/reviewed the manuscript with help of M.J.R. as part of her Doctorate of Science in Chemical Engineering. All authors have given approval to the final version of the manuscript. Funding: This work was financed by National Council for Scientific and Technology Development (CNPq, grant 483947/2012-1, and doctorate fellowship of M.J.R.), and in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES), Finance Code 001. We also gratefully recognize all support from the Project MINECO from Spanish Government, (project number CTQ2017-86170-R). Acknowledgments: The authors thank LNF Latin America (Bento Gonçalves, RS, Brazil) for providing the amylolytic enzymes (Toryzyme®3.0L and BAN®480L). Conflicts of Interest: The authors declare no conflicts of interest.

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