A Green and Simple Protocol for Extraction and Application of a Peroxidase-Rich Enzymatic Extract

Protocol A Green and Simple Protocol for Extraction and Application of a Peroxidase-Rich Enzymatic Extract

Gonçalo P. Rosa 1 , Maria do Carmo Barreto 1 , Diana C. G. A. Pinto 2 and Ana M. L. Seca 1,2,*

1 cE3c—Centre for Ecology, Evolution and Environmental Changes/Azorean Biodiversity Group & Faculty of Sciences and Technology, University of Azores, Rua Mãe de Deus, 9500-321 Ponta Delgada, Portugal; [email protected] (G.P.R.); [email protected] (M.d.C.B.) 2 LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; [email protected] * Correspondence: [email protected]; Tel.: +351-296-650-74

Received: 7 February 2020; Accepted: 24 March 2020; Published: 26 March 2020

Abstract: Recently there is a great social expectation that scientists should produce more sustainable and environmentally friendly chemical processes. Within this necessity, biocatalysis presents many attractive features because reactions are often performed in water, under mild conditions, the catalyst is biodegradable and can be obtained from renewable raw materials. In this work, we propose a simple, rapid and low-cost method for the preparation and application of an enzymatic extract from turnip root. The protocol described includes (1) the preparation of the enzymatic extract, (2) the procedure for the assessment of the more favorable working parameters (temperature, pH) and (3) the methodology for the application of the extract as the catalyst for biotransformation reactions. We anticipate that the protocol in this research will provide a simple way for obtaining an enzymatic extract which can operate eﬃciently under mild conditions and can eﬀectively catalyze the biotransformation of simple phenols.

Keywords: Brassica rapa; extraction; enzymatic extract; biocatalysis; peroxidase

1. Introduction Recently, there is a higher societal awareness regarding the environmental questions of human activities, and science is no exception [1]. This increasing expectation that scientists should improve processes to become greener and more sustainable, led to the development of green chemistry. This term, postulated by Anastas and Warner [2], aims toward developing new products and processes that are less dangerous to human health and the environment, by eliminating or reducing the use and production of harmful substances as well as reducing harmful or toxic intermediates [3]. Basic principles of green chemistry cover a wide spectrum of organic synthesis methodology such as designing processes of organic synthesis to reduce byproduct/waste generation, reduce the use of hazardous chemicals/raw materials and enhance the use of safer or more environmentally-safe solvents and renewable raw materials. In addition, green chemistry promotes the best form of waste treatment, designing the process of chemical residues degradation, all following pollution prevention and sustainable development procedures, also focusing on energetic efficiency [4]. Within these principles comes catalysis, which has many attractive features in the context of green chemistry. Catalysts and their associated catalytic reactions could be homogeneous, heterogeneous or use enzymatic (bio-) catalysis. In homogeneous catalysis, the catalyst and the reactants are in the same phase [5]. Homogeneous catalysts apply to reactions in the gas phase or mostly in the liquid phase. Although these catalysts have some advantages such as excellent selectivity and single active sites, they suffer from some disadvantages, like poor thermal stability and difficult and high cost catalyst

Methods Protoc. 2020, 3, 25; doi:10.3390/mps3020025 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 25 2 of 12 recovery [5]. Heterogeneous catalysts typically involve the use of solid catalysts placed in a liquid or gas reaction mixture [6]. Since they are in a different phase, heterogeneous catalysts can be easily separated from a reaction mixture, allowing the effective recovery and recycling of the catalyst, which is quite eco-friendly. Furthermore, this type of catalysts is very stable, maintaining the activity even under harsh reaction conditions [6]. Despite such advantages, heterogeneous catalysis has been thought to allow for less interaction and controllability than homogeneous catalysis due to a poor understanding of its reaction process [6]. With biocatalysts, the reactions are often performed in water, under mild conditions of temperature, pressure and pH [7,8]. Furthermore, the catalyst (an enzyme) is biodegradable and could be derived from renewable raw materials. The enzyme often affords high chemo-, regio- and stereoselectivities and avoids the need for protection and deprotection sequences which are required in classical synthesis methodology [1,9]. Some factors limit the industrial applications of enzymes, namely low long-term operational stability, in addition to the technically challenging process of recovery and reuse of the enzyme. In order to render enzyme utilization in biotechnological processes more favorable, different methods for cost reduction have been put into practice, one of which is immobilization [10]. The term “immobilized enzymes” refers to “enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously” [11]. In addition to a more convenient handling of the enzyme, it also substantially simplifies the control of the reaction process [12] while enhancing the stability of the enzyme under both storage and operational conditions. Immobilization provides also an easy separation of the enzyme from the product, hence protein contamination of the product is minimized or even avoided [13]. Apart from simplifying the separation of the enzyme from the reaction mixture, enzyme immobilization in a matrix, like alginate, also remarkably reduces the cost of enzyme and consequently of the enzymatic products [10], since it usually allows using the enzyme for a longer period. However, enzymatic immobilization presents also some disadvantages like lower enzyme activity compared to native enzyme, additional costs with the matrix used for immobilization and lower reaction rates compared to native enzymes [14]. Biotransformation of chemical compounds usually resorts to using unpurified enzymatic extracts, since the associated cost is not too high, although these extracts only contain approximately 1%–30% of the pretended enzyme, besides other active and/or inactive enzymes and cell constituents. Additionally, enzymatic extracts are often more stable than purified enzymes due to the presence of a mixture of components that confer protection against undue oxidation and denaturation [7]. A group of enzymes often used in these procedures are peroxidases, which, due to their versatility and to their ubiquitous nature, have immense potential to be employed as industrial enzymes [15]. They catalyze the redox reaction for a wide range of substrates, which leads to the classification of peroxidases as an important group of enzymes for medicinal, biochemical, immunological, biotechnological and industrial applications. They have been successfully used for biopulping and biobleaching in the paper and textile industries [16,17]. Additionally, peroxidases extracted from different sources have been used for bioremediation and decolorization reactions [18–21]. They have been able to efficiently remove phenol derivatives from wastewater, like phenol, 2-chlorophenol, m-cresol, by oxidative polymerization, with 95% removal efficiency when associated with polyethylene glycol, and without needing further purification of the extract, which greatly reduces the associated costs [21]. Purified peroxidases have also been used in organic synthesis, bioremediation, as well as various analytical applications. For example, peroxidase-based biosensors find application in analytical systems for determination of hydrogen peroxide, glucose, alcohols, glutamate and choline [22]. Interesting works on the production of recombinant peroxidases in several hosts have been published so far, searching for a way to obtain a homogeneous and high yield source of these enzymes. Nevertheless, it has to be pointed out that the production of these recombinant enzymes still has not achieved the desired yields [23]. In Escherichia coli, the host where higher yields have been obtained, the enzymes are often found as protein aggregates which require laborious solubilization and refolding steps in order to obtain activity, with yields that make this production not competitive [24]. For most Methods Protoc. 2020, 3, 25 3 of 12 biotechnological and industrial purposes, therefore, the use of vegetable peroxidase sources is so far the more economic option. In this regard, the present work aims to present a methodology for the simple, rapid and low-cost preparation of a peroxidase-rich enzymatic extract suitable not only for application in biotransformation reactions but also for application for educational purposes. In fact, the proposed protocol can be used in laboratory classes so that students acquire knowledge on how to obtain an enzyme extract and understand the concept of enzyme activity and some of the factors on which that activity depends.

2. Experimental Design The protocol in this study is designed for the rapid preparation of a peroxidase-rich enzymatic extract from turnip roots (Brassica rapa var. rapa Barkant), obtained from a local market, which will be ready to be applied as a catalyst in biotransformation reactions. The turnip root was chosen due to availability, but the protocol described can be used for other peroxidase sources [25–29]. In a ﬁrst stage, the steps for obtaining the extract are presented, then the method for determining the extract’s enzymatic activity and optimizations are discussed, and ﬁnally, the procedure for the applying the extract as a catalyst in a biotransformation of guaiacol is explained.

2.1. Materials

2.1.1. Preparation of Enzymatic Extract Fresh turnip (Brassica rapa var. rapa Barkant) roots. • Phosphate buffer 100 mM pH 6.5. • Cotton wool. • 2.1.2. Protein Quantification Bradford Reagent • Bovine Serum Albumin (BSA) (Sigma, Darmstadt, Germany) • 2.1.3. Biotransformation Reaction 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma, • 0 Darmstadt, Germany). Phosphate buffer 300 mM pH 5.5, 6.5 and 7.5. • Acetate buffer 300 mM pH 3.5 and 4.5. • Hydrogen peroxide (H O ) (Fluka, Charlotte, NC, USA). • 2 2 Dimethyl sulfoxide (DMSO) (Sigma, Darmstadt, Germany). • Acetone (Sigma, Darmstadt, Germany). • Guaiacol (2-methoxyphenol) (Sigma, Darmstadt, Germany). • 2.2. Equipment Eppendorf 5810R centrifuge (Eppendorf—Hamburg, Germany). • EVOLUTION 160 UV–Vis Thermo Scientific thermostated spectrophotometer (Thermo Fisher • Scientific, Waltham, MA, USA).

3. Procedure

3.1. Preparation of Enzymatic Extract. Time for Completion: 00:15 h 1. Wash and peel the turnip roots. 2. Dice 20 g of the peeled turnip roots. 3. Add 100 mL of cold phosphate buﬀer 100 mM, pH 6.5 to the diced roots. Methods Protoc. 2020, 3, 25 4 of 12

4. Homogenize the mixture, immersed in ice using a commercial blender, until no lumps are observed. 5.Methods Filter Protoc. the 20 homogenized20, 3, x FOR PEER mixture REVIEW with cotton wool to remove suspended ﬁbrous solid particles.4 of 12 6. Centrifuge the ﬁltrate at 18,514 g for 5 min at 4 C. × ◦ 7.6. CollectCentrifuge the supernatant the filtrate at and 18, store514 x it g infor ice. 5 min at 4 °C. 7. Collect the supernatant and store it in ice. PAUSE STEP IfIf thethe extractextract preparedprepared isis not not going going to to be be readily readily used, used, it it can can be be stored stored at at 80−80 C°C − ◦ withoutwithout lossloss of activity.

3.2.3.2. Protein Protein Quantiﬁcation—Bradford Quantification—Bradford Assay. Assay. Time Time for for Completion: Completion: 00:30 00:30 h h ThisThis assayassay waswas performed performed based based on on Bradford Bradford test test [ 30[30].].

1.1. PreparePrepare BSA BSA dilutions dilutions for for standards standards (from (from 100–1000 100–1000µ gµg/mL)./mL). 2.2. UseUse phosphate phosphate bu bufferffer 100 100 mM, mM, pH pH 6.5 6.5 as as a a blank blank (0 (0µ µg/mL).g/mL). 3. Mix 2500 µL of Bradford protein-dye solution with standards or enzymatic extract samples. 3. Mix 2500 µL of Bradford protein-dye solution with standards or enzymatic extract samples. Methods4. Incubate Protoc. 20 for20, 3 10, x FORmin. PEER REVIEW 5 of 12 4. Incubate for 10 min. 5. Measure the absorbance at 595 nm. 5. Measure the absorbance at 595 nm. 2.6. PreparePlot a concentration/absorbance 20 mL of substrate at a curveconcentration with the ofvalues 7.25 obtainedmM in acetate for standards. buffer 300 mM (pH 4.5). 6. Plot a concentration/absorbance curve with the values obtained for standards. 7. OtherSubstitute volumes the absorbance can be used, values if needed. of the extract samples on the equation of the curve to obtain 7. Substitutethe total protein the absorbance concentration. values of the extract samples on the equation of the curve to obtain the totalOPTIONAL protein concentration.STEP If the substrate is not soluble under these conditions, prepare the substrate with an appropriate solvent and add the buffer to obtain a mixture where the enzymatic extract 3.3. Biotransformation Reaction. 3.3. Biotransformationretains if not all, Reaction at least a % of its activity. This proportion can be assessed by the procedure described in Section 3.3.1. 3.3.1.3.3.1. Assessment Assessment of of Extract extract Activity. activity. TimeTime forfor Completion:completion: 00:05 h 3. Gradually add 0.4 mL of H2O2 (9.43 mM) to the reaction mixture every 8 h, to a final volume of 1. On two 3 mL cuvettes, add 2150 μL of acetate buffer 300 mM (pH 4.5), each. 1. On2.4 twomL. 3 mL cuvettes, add 2150 µL of acetate buffer 300 mM (pH 4.5), each. CRITICAL STEP All reagents and samples need to be brought to 35 °C in advance, to prevent temperature CRITICALCRITICAL fluctuations STEPSTEP AllThe reagentsduring concentration the and test. samples of H2O need2 is higher to be broughtthan substrate to 35 ◦C concentration in advance, to to prevent prevent temperatureH2O2 from becoming fluctuations a limiting during step. the test. The addition of this reactive should be performed in small 2. Proceed to add 100 μL ABTS (0.7 mM in acetate buffer 300 mM pH 4.5) to both cuvettes. 2. Proceedportions toduring add 100 theµ reaction,L ABTS (0.7rather mM than in acetate adding bu allff erin 300the mMbeginning. pH 4.5) This to both is due cuvettes. to the fact that addingOPTIONAL a large STEP amount Other at colored once may substrates cause thecan decompositionbe used, as long of as thethey p reactrosthetic with heme the enzymatic and also OPTIONAL STEP Other colored substrates can be used, as long as they react with the enzymatic modificationextract, resulting of the in highera change structure in absorbance. of peroxidase, both essential for enzyme activity [31]. extract, resulting in a change in absorbance. OPTION Other time intervals for adding H2O2 can be chosen, depending on the laboratory 3.3. AddAdd 5050 µμLL ofof H 2O2 0.30.3% %(v/v) (v/v) in in acetate acetate buffer buffer 300 300 mM mM (pH (pH 4.5) 4.5) to both to both cuvettes cuvettes and andmix mixthem equipment to ensure2 2 the gradual and fractionated addition of the reactive, like peristaltic pumps. themthoroughly. thoroughly. 4. StartPut the the cuvette reaction in by the adding thermostated 750 μL ofspectrophotometer, turnip enzymatic extractwith the and temperature repeat every set 12 to h, 35 to °C, ensure and 4. Put the cuvette in the thermostated spectrophotometer, with the temperature set to 35 ◦C, and let itthatlet remain it there remain there are there always for 2–3 for active min,2–3 min, to molecules reach to reach temperature of temperature the enzyme equilibrium. equ in theilibrium. reaction medium. 5. To the first cuvette, add 50 μL of acetate buffer 300 mM (pH 4.5), and set the blank in the 5. ToOPTION the first The cuvette, addition add of 50 enzymaticµL of acetate extract bu andffer H 3002O2 mM can (pHbe done 4.5), simultaneously, and set the blank but in it thealso spectrophotometer, at 414 nm, with the temperature set to 35 °C. spectrophotometer,depends on laboratory at 414 equipment. nm, with theIn fact, temperature in this work set to the 35 ◦conditionsC. chosen were limited by 6. To the remaining cuvette, add 50 μL of the enzymatic extract and shake the mixture. 6. Toonly the one remaining peristaltic cuvette, pump add with 50 aµ timer,L of the used enzymatic to the hydrogen extract and peroxide shake theaddition, mixture. while enzyme 7. Immediately after the addition of the extract, start registering the absorbance in the thermostated 7. Immediatelyaddition was after performed the addition manually. of the extract, Considering start registering that in the the blank absorbance reaction, in the performed thermostated with spectrophotometer, always with the temperature set to 35 °C. spectrophotometer,peroxide but not enzymatic always with extract, the temperature there were set no to 35 changes◦C. in the reactional medium, the conditions CRITICAL chosen STEP did notTo affect obtain the a development more correct of assessmentthe catalytic ofreaction. the enzymatic activity, the absorbanceCRITICAL has STEPto be Toregistered obtain a morefrom correctthe moment assessment the reaction of the enzymatic starts, so activity,step 6 must the absorbance be done in 5. Stop the reaction after 48 h. hasthe fastest to be registered way possible. from the moment the reaction starts, so step 6 must be done in the fastest way possible. 4.8. ExpectedFollow Resultsthe absorbance of the sample at 414 nm, for 90 s. 8. Follow the absorbance of the sample at 414 nm, for 90 s. 9. With the data obtained, plot a curve which will allows calculating the enzymatic activity of 9.4.1. WithPropertiesthe extract. the dataof Enzymatic obtained, Extract plot a curve which will allows calculating the enzymatic activity of the extract. OPTIONALThe described STEP extraction All the processreaction isparameters a very simple (temperature, procedure pH, to etc.) obtain described a ready are-to -optimaluse turnip for enzymaticOPTIONALthis enzymatic extract. STEP However,extractAll theactivity reactionit is importantto the parameters substrate to assess (temperature, ABTS. some Ho wever, properties pH, etc.)if there ofdescribed this is the extract. areneed optimal Thus,to test forit the is necessarythiseffect enzymatic to of verify changing extract if it is, these activityin fact, parameters an to theextract substrate in that the exhibits ABTS. activity However, enzymatic of the enzymatic if activity, there is the extractto determine need or, to test if what a the different e ffisect the proteinsubstrate content is presentused, the and working the variability parameters of these could parameters be reassessed between using extractions.the same methodology used withThe resultsABTS. show that, besides being a quite fast procedure, it also yielded an extract with good amounts of protein per mL, and good total peroxidase activity (Table 1). In fact, when compared with similar3.3.2. Biotrans works formation.from the literature, Time for completion:the presented 48:00 method h has the advantage of being the one where the extract is obtained in fewest time, with more activity and with less intermediate steps [32,33]. 1. Set a water bath to 35 °C. The total protein values determined were virtually unchanged, in various extractions performed with roots obtained in autumn. For turnip roots obtained in the winter, the total amount of protein present in the extract was much lower (Table 1).

Methods Protoc. 2020, 3, 25 5 of 12

of changing these parameters in the activity of the enzymatic extract or, if a diﬀerent substrate is used, the working parameters could be reassessed using the same methodology used with ABTS.

3.3.2.Methods Biotransformation. Protoc. 2020, 3, x FOR TimePEER REVIEW for Completion: 48:00 h 5 of 12

1. Set a water bath to 35 ◦C. 2. Prepare 20 mL of substrate at a concentration of 7.25 mM in acetate buffer 300 mM (pH 4.5). 2. Prepare 20 mL of substrate at a concentration of 7.25 mM in acetate buﬀer 300 mM (pH 4.5). Other Other volumes can be used, if needed. volumes can be used, if needed. OPTIONAL STEP If the substrate is not soluble under these conditions, prepare the substrate OPTIONAL STEP If the substrate is not soluble under these conditions, prepare the substrate with an appropriate solvent and add the buffer to obtain a mixture where the enzymatic extract with an appropriate solvent and add the buﬀer to obtain a mixture where the enzymatic extract retains if not all, at least a % of its activity. This proportion can be assessed by the procedure retains if not all, at least a % of its activity. This proportion can be assessed by the procedure described in Section 3.3.1. described in Section 3.3.1.

3.3. GraduallyGradually add add 0.4 0.4 mL mL of of H H2O2O22(9.43 (9.43 mM)mM) toto thethe reactionreaction mixture mixture every every 8 8 h, h, to to a a ﬁnal final volume volume of of 2.42.4 mL. mL.

CRITICALCRITICAL STEPSTEPThe The concentrationconcentration of of H H2O2O22is is higherhigher thanthan substrate substrate concentration concentration to to prevent prevent HH22O2 fromfrom becoming becoming a limiting step.step. TheThe addition ofof thisthis reactivereactive shouldshould be be performed performed in in small small portionsportions duringduring thethe reaction,reaction, ratherrather thanthan addingadding all all in in the the beginning. beginning. This This is is due due to to the the fact fact that that addingadding aa largelarge amountamount atat onceonce maymay causecause thethe decomposition decomposition ofof the the prosthetic prosthetic heme heme and and also also modiﬁcationmodification ofof thethe higherhigher structurestructure ofof peroxidase,peroxidase, bothboth essentialessential forfor enzymeenzyme activityactivity [[31].31]. OPTION Other time intervals for adding H2O2 can be chosen, depending on the laboratory OPTION Other time intervals for adding H O can be chosen, depending on the laboratory equipment to ensure the gradual and fractionated2 2 addition of the reactive, like peristaltic pumps. equipment to ensure the gradual and fractionated addition of the reactive, like peristaltic pumps. 4.4. StartStart the the reaction reaction by by adding adding 750 750µ μLL of of turnip turnip enzymatic enzymatic extract extract and and repeat repeat every every 12 12 h, h, to to ensure ensure thatthat there there are are always always active active molecules molecules of of the the enzyme enzyme in in the the reaction reaction medium. medium.

OPTIONOPTION TheThe additionaddition ofof enzymaticenzymatic extractextract andand H H2O2O22 cancan bebe donedone simultaneously, simultaneously, but but it it also also dependsdepends onon laboratory laboratory equipment. equipment. In In fact, fact, in thisin this work work the conditionsthe conditions chosen chosen were were limited limited by only by oneonly peristaltic one peristaltic pump pump with a with timer, a usedtimer, to used the hydrogen to the hydrogen peroxide peroxide addition, addition, while enzyme while addition enzyme wasaddition performed was pe manually.rformed Consideringmanually. Considering that in the blank that inreaction, the blank performed reaction, with performed peroxide butwith notperoxide enzymatic but extract, not enzymatic there were extract, no changes there in were the reactional no changes medium, in the the reactional conditions medium, chosen did the notconditions aﬀect the chosen development did not affect of the the catalytic development reaction. of the catalytic reaction. 5.5. StopStop the the reaction reaction after after 48 48 h. h.

4.4. Expected Expected Results Results 4.1. Properties of Enzymatic Extract 4.1. Properties of Enzymatic Extract The described extraction process is a very simple procedure to obtain a ready-to-use turnip The described extraction process is a very simple procedure to obtain a ready-to-use turnip enzymatic extract. However, it is important to assess some properties of this extract. Thus, it is enzymatic extract. However, it is important to assess some properties of this extract. Thus, it is necessary to verify if it is, in fact, an extract that exhibits enzymatic activity, to determine what is the necessary to verify if it is, in fact, an extract that exhibits enzymatic activity, to determine what is the protein content present and the variability of these parameters between extractions. protein content present and the variability of these parameters between extractions. The results show that, besides being a quite fast procedure, it also yielded an extract with good The results show that, besides being a quite fast procedure, it also yielded an extract with good amounts of protein per mL, and good total peroxidase activity (Table1). In fact, when compared with amounts of protein per mL, and good total peroxidase activity (Table 1). In fact, when compared with similar works from the literature, the presented method has the advantage of being the one where the similar works from the literature, the presented method has the advantage of being the one where extract is obtained in fewest time, with more activity and with less intermediate steps [32,33]. the extract is obtained in fewest time, with more activity and with less intermediate steps [32,33]. The total protein values determined were virtually unchanged, in various extractions performed The total protein values determined were virtually unchanged, in various extractions performed with roots obtained in autumn. For turnip roots obtained in the winter, the total amount of protein with roots obtained in autumn. For turnip roots obtained in the winter, the total amount of protein present in the extract was much lower (Table1). present in the extract was much lower (Table 1).

Methods Protoc. 2020, 3, x FOR PEER REVIEW 6 of 12 Methods Protoc. 2020, 3, 25 6 of 12 Table 1. Total amount of protein present on the enzymatic extract obtained from turnip roots in different seasons. Table 1. Total amount of protein present on the enzymatic extract obtained from turnip roots in Total Activity different seasons.Extraction Season Total Protein (μg/mL Extract) (U/μg Protein) Extraction1 SeasonAutumn Total Protein (µg349/mL ± Extract)11 Total Activity0.77 ± 0.05 (U/ µg Protein) 2 Autumn 358 ± 15 0.81 ± 0.02 1 Autumn 349 11 0.77 0.05 3 Autumn ± 353 ± 26 0.79 ± 0.06± 2 Autumn 358 15 0.81 0.02 ± ± 34 Autumn Autumn 353 35126 ± 18 0.80 0.79 ± 0.030.06 ± ± 45 Autumn Winter 351 18201 ± 6 0.54 0.80 ± 0.010.03 ± ± 56 WinterWinter 201 2056 ± 6 0.57 0.54 ± 0.050.01 ± ± 67 WinterWinter 205 2116 ± 13 0.60 0.57 ± 0.020.05 ± ± 78 WinterWinter 211 13210 ± 4 0.59 0.60 ± 0.010.02 ± ± 8 Winter 210 4 0.59 0.01 ± ± These variations in total protein concentration were reflected in the total activity (U) of the extract,These with variations the extracts in total prepared protein with concentration roots collected were in reflected winter, in being the total less activityactive than (U) ofthe the ones extract, with withthe roots the extracts collected prepared in autumn with roots(Table collected 1). This invariabil winter,ity being has lessbeen active described than thefor onesturnip with peroxidase, the roots collectedwhere it was in autumn found that (Table the1). concentration This variability and hasactivity been of described this enzyme for turnipvaries seasonally, peroxidase, depending where it wason the found development that the concentration stage [34]. In this and regard, activity it ofis recommended this enzyme varies that large seasonally, amounts depending of extract onshould the developmentbe prepared in stage seasons [34]. where In this it regard,is more itactive is recommended and, stored at that −80 large °C to amounts further usage. of extract should be prepared in seasons where it is more active and, stored at 80 ◦C to further usage. 4.2. Assessment of Extract Activity in Different Conditions − 4.2. Assessment of Extract Activity in Different Conditions The assessment of extract activity in different conditions is important for the determination of whichThe conditions assessment are of more extract suitable activity for in the di ffbiotransformationerent conditions isreactions, important and for also, the determinationin case that some of whichproblems conditions appear are during more suitable the application for the biotransformation on the biotransformation reactions, and reaction, also, in to case verify that whichsome problemsadjustments appear can duringbe made, the without application deeply on the affecting biotransformation the activity of reaction, the enzymatic to verify extract which. adjustments can beIn made, this stage, without it is deeply possible affecting to test the the activity optimal of temperature, the enzymatic pH, extract. the stability of the enzymatic extractIn thiswhen stage, exposed it is possible to certain to testtemperatures the optimal for temperature, long periods pH, or the the stability effect of of solvents the enzymatic in its activity. extract whenOnly exposed with this to certain information temperatures it is for possible long periods to choose or the etheffect most of solvents suitable in its parameters activity. Only for with the thisbiotransformation information it is reaction. possible to choose the most suitable parameters for the biotransformation reaction. TheThe firstfirst parameter parameter to to be be tested tested was was the the storage storage stability stability of of the the extract, extract, because because of seasonalof seasonal loss loss of proteinof protein content content and activity and activity reported reported above. above. The extract The extract was stored was at stored80 C at and−80 its °C activity and its assessed activity − ◦ everyassessed 7 days every until 7 thedays 35th until day. the The 35th extract day. retainedThe extract full retained activity after full 35activity days, after and it35 was days, observed and it thatwas duringobserved that that period during of storage, that period namely of betweenstorage, thenamely 7th and between the 21th the day, 7ththe and extract the 21 activityth day, wasthe higherextract thanactivity the was registered higher in than day the 0 (Figure registered1). in day 0 (Figure 1).

FigureFigure 1.1. CurveCurve ofof enzymaticenzymatic extractextract stabilitystability whenwhen storedstored atat −8080 °C;C; pHpH4.5. 4.5. ErrorError barsbars representrepresent − ◦ standardstandard deviation. deviation.

ThisThis is is most likely likely because because the the freezing freezing process process destroys destroys some some cellular cellular structures structures that werethat werepreserved preserved during during the ex thetraction extraction process, process, which couldwhich be could encasing be encasing part of the part peroxidase, of the peroxidase, therefore contributing to a higher availability of the free enzyme and consequently to an increased reaction rate.

Methods Protoc. 2020, 3, 25 7 of 12 therefore contributing to a higher availability of the free enzyme and consequently to an increased Methods Protoc. 2020, 3, x FOR PEER REVIEW 7 of 12 reaction rate. The optimal operating pH was found to be pH 4.5, with an activity of 0.772 UU/µg/µg protein. The other pH valuesvalues testedtested yieldedyielded significantly significantly lower lower activities, activities, with with the the lowest lowest activity activity being being obtained obtained at pHat pH 3.5. The3.5. The low low activity activity obtained obtained at pHat pH 3.5 3.5 might might be be related related with with enzyme enzyme inactivation inactivationcaused caused byby thethe acidic conditions, since low pH usually causes the the denaturation denaturation of of proteins, proteins, leading leading to to their their loss loss of of function. function. There was no significant significant difference difference between between the the enzyme enzyme activity activity at at pH pH 5.5 5.5 and and 6.5 6.5 (Figure (Figure 2).2).

FigureFigure 2. 2.E Effectﬀect ofof pHpH onon enzymaticenzymatic extract activity at 30 °C.◦C. Error Error bars bars represent represent standard standard deviation. deviation.

Regarding temperature, the maximum activity activity was was achieved achieved at at 50 50 °C◦C with with 1.416 1.416 U/µg U/µg protein, protein, although the difference diﬀerence of activity from 4545 toto 6060 ◦°CC waswas not tootoo pronouncedpronounced (Figure(Figure3 3).).

Figure 3. EffectEﬀect of of temperature on enzymatic extract activity at at pH pH 4.5. 4.5. Error Error bars represent standard deviation.

The optimal temperature for the extract activity was 10 °C◦C higher than the one obtained by Motamed et al. [32]. [32]. This This may may be be due due to to the the fact fact that that in in the cited work the enzyme was was semi semi-pure,-pure, and in this work, the enzyme was in an extract extract together together with other constituents. The The presence presence of other constituents protects the enzyme against denaturation, which may explain wh whyy the enzymatic extract resisted higher temperatures than the semi-puresemi-pure enzymeenzyme studiedstudied byby MotamedMotamed etet al.al. [[32].32]. To assess assess the the stability stability of of the the extract extract over over long-term long-term exposure exposure to temperature, to temperature, the enzymatic the enzymatic extract wasextract incubated was incubated during during 72 h at 72 di hﬀ erentat different temperatures, temperatures, with thewith activity the activity being being assessed assessed every every 24 h. After24 h. 24 After h, the 24 activity h, the activityof the extract of the was extract almost was null almost (data are null not (data shown) are atnot the shown) higher at temperatures. the higher temperatures. Even when incubated at 40 °C, a severe activity loss was seen after 24 h, while at 30 °C the tendency for the activity to decrease only became most significant after 48 h (Figure 4).

Methods Protoc. 2020, 3, 25 8 of 12

Even when incubated at 40 ◦C, a severe activity loss was seen after 24 h, while at 30 ◦C the tendency for the activity to decrease only became most signiﬁcant after 48 h (Figure4). MethodsMethods Protoc.Protoc. 20202020,, 33,, xx FORFOR PEERPEER REVIEWREVIEW 88 ofof 1212

FigureFigureFigure 4. 4.4.Curve CurveCurve of ofof long-term longlong--termterm enzymatic enzymaticenzymatic extract extractextract thermostability thermostabilitythermostability for 72 forfor h, 7272 at pH:h,h, atat 4.5 pH:pH: and 4.54.5 30 andand◦C (left 3030 °C°C panel) (left(left orpanel)panel) 40 ◦C oror (right 4040 °C°C panel). (right(right panel). Errorpanel). bars ErrorError represent barsbars representrepresent standard standardstandard deviation. deviatdeviationion..

WithWithWith these these these results, results, results, it it it became became became evident evident evident that that that the the the enzymatic enzymatic enzymatic extract extract extract would would would not not not remain remain remain active active active in in in longer-termlongerlonger--termterm reactions reactionsreactions at atat 40 4040◦ °C.°C.C. However, However,However, the thethe enzymatic enzymaticenzymatic extract extractextract activity activityactivity is isis lower lowerlower at atat 30 3030◦ °C,°C,C, as asas observable observableobservable ininin Figure FFigureigure3 . 3. 3. In In In this this this regard, regard, regard, the the the temperature temperature temperature of of of 35 35 35 ◦ °C °CC waswaswas elected elected elected the the the most most most suitable suitable suitable for for for long-time long long--timetime reactions,reactions,reactions, in inin an anan attempt attemptattempt to toto maintain maintainmaintain a aa compromise compromisecompromise between betweenbetween extract extractextract activity activityactivity and andand its itsits stability. stability.stability. Sometimes,Sometimes,Sometimes, the thethe intended intendedintended substrate substratesubstrate is isis not notnot soluble solublesoluble in inin the thethe bu bufferbufferffer used usedused as asas a aa reaction reactionreaction medium mediummedium for forfor the thethe enzyme.enzyme.enzyme. In InIn this thisthis case, case,case, it itit might mightmight be bebe necessary necessarynecessary to toto prepare prepareprepare the thethe substrate substratesubstrate with withwith an anan appropriate appropriateappropriate solvent solventsolvent and andand addaddadd the thethe bu bufferbufferffer to toto obtain obtainobtain a aa mixture mixturemixture where wherewhere the thethe enzymatic enzymaticenzymatic extract extractextract retains retainsretains if ifif not notnot all, all,all, at atat least leastleast a aa significant significantsignificant fractionfractionfraction of ofof its itsits total totaltotal activity. activity.activity. In InIn the thethe specific specificspecific case casecase of ofof the thethe turnip turnipturnip enzymatic enzenzymaticymatic extract, extract,extract, it itit retains retainsretains about aboutabout 40% 40%40% of ofof itsitsits activity activityactivity when whenwhen exposed exposedexposed to toto a aa concentration concentrationconcentration of ofof 20% 20%20% (v (v/v)(v/v)/v) DMSO DMSODMSO (Figure (Figure(Figure5 ,5,5, left leftleft panel) panel)panel) and andand 35% 35%35% when whenwhen exposedexposedexposed to toto same samesame concentration concentrationconcentration of ofof acetone acetoneacetone (Figure (Figure(Figure5 ,5,5, right rightright panel), panel),panel), so soso either eithereither one oneone of ofof these thesethese solvents solventssolvents could couldcould bebebe selected selectedselected depending ddependingepending on onon substrate substratesubstrate solubility, solubility,solubility, without withoutwithout complete completecomplete loss lossloss of ofof activity. activity.activity.

FigureFigureFigure 5. 5.5.E EffectEffectﬀect of ofof DMSO DMSODMSO (left (left(left panel) panel)panel) or oror acetone acetoneacetone (right (right(right panel) panel)panel) in inin enzymatic enzymaticenzymatic extract extractextract activityactivityactivity atatat pHpHpH 4.5.4.5.4.5. ErrorErrorError bars barsbars represent representrepresent standard standardstandard deviation. deviationdeviation..

4.3.4.3.4.3. Biotransformation BiotransformationBiotransformation Reaction ReactionReaction ToToTo demonstrate demondemonstratestrate the thethe expected expectedexpected results resultsresults when whenwhen the thethe enzymatic enzymaticenzymaticextract extractextract is isis applied appliedapplied on onon biotransformation biotransformationbiotransformation reactionsreactionsreactions performed performedperformed as asas described describeddescribed in pointinin pointpoint Section 3.3.2,3.3.2, 3.3.2 itit waswas, it was selectedselected selected thethe the phenolicphenolic phenolic compoundcompound compound guaiacol,guaiacol, guaiacol, aa asubstratesubstrate substrate that that that had had had been been been previously previously biotransformed biotransformed with with a a peroxidase peroxidase enzyme en enzymezyme [[35][35]35],,, which which which would would would allowallowallow a aa better betterbetter assessment assessmentassessment of ofof the thethe e efficacyefficacyﬃcacy of ofof the thethe enzymatic enzymaticenzymatic extract extractextract obtained obtainedobtained by byby the thethe proposed proposedproposed protocol. protocol.protocol. AccordingAccordingAccording to to to the the the literature, literature, literature, the the the reaction reaction reaction of of of guaiacol guaiacol guaiacol with with with hydrogen hydrogen hydrogen peroxide peroxide peroxide catalyzed catalyzed catalyzed by by by a a a peroxidaseperoxidaseperoxidase should shouldshould lead leadlead to toto the thethe formation forformationmation of ofof tetraguaiacol tetraguaiacoltetraguaiacol (Figure (Figure(Figure6 (1a)), 66 1a1a),), a aa tetramer tetramertetramer of ofof guaiacol. guaiacol.guaiacol.

Methods Protoc. 2020, 3, 25 9 of 12 Methods Protoc. 2020, 3, x FOR PEER REVIEW 9 of 12

Figure 6. PeroxidasePeroxidase-catalyzed-catalyzed transformation of guaiacol ( 1) to tetraguaiacol tetraguaiacol ( 1a).

The biotransformation biotransformation of of guaiacol guaiacol (Figure (Figure 6 16)(1)) catalyzed catalyzed by the by enzymatic the enzymatic extract extract in the inchosen the reactionalchosen reactional conditions conditions (Figure (Figure 6) occurred6) occurred very very quickly quickly and and is is easily easily observable observable even even without without spectrophotometric methods, as seen by the shift of color from transparenttransparent toto amberamber (Figure(Figure7 7).).

Figure 7. Biotransformation reaction: reaction: Development Development of of the the reaction reaction using using gua guaiacoliacol catalyzed by the extractextract.. A (A A)t At 0 min; 0 min; B (atB )5 at min 5 min;; C 10 (C min.) 10 min.

A blank reaction was also performed, in the same conditions, conditions, but with the the addition addition of of phosphate phosphate bufferbuffer instead of the enzymatic extract. In In this this reaction reaction no no transformation transformation was was observed, observed, whic whichh shows that thethe enzymaticenzymatic extract extract was was definitively definitively responsible responsible for for the observedthe observed transformation transformation and notand a resultnot a resultof the of direct the direct reaction reaction between between the guaiacol the guaiacol and the and H2 theO2. H2O2. Since changes changes in in the the chemical chemical structure structure of phenolic of phenolic compounds compounds are often are reflected often reflected in the UV/Vis in the absorptionUV/Vis absorption spectra, the spectra, reaction the progression reaction progression was followed was by followed recording by the recording absorption the spectrum absorption of reactionalspectrum of mixture reactional over mixture reaction over time. reaction The time. reaction The progressio reaction progressionn can be also can be followed also followed by other by methodsother methods like zymography, like zymography, high high performance performance liquid liquid chromatography chromatography (HPLC (HPLC)) or or nearnear-infrared-infrared spectroscopy [29,35,36],[29,35,36], however, however, since since the the main main goal goal of this of this protocol protocol is to is describe to describe a simple a simple and low and- costlow-cost method, method, the authors the authors opted opted for UV/Vis for UV /spectrophotometry.Vis spectrophotometry. As described in the literature [[36],36], thethe tetraguaiacoltetraguaiacol (Figure(Figure6 6(1a)) 1a) absorbsabsorbs radiationradiation betweenbetween 400400 and 500 500 nm nm and and has has its maximum its maximum absorbance absorbance at 470 at nm. 470 Thus, nm. the Thus, absorbance the absorbance level at this level wavelength at this wavelengthover the reaction over the time reaction is used time as a markeris used ofas guaiacola marker peroxidation. of guaiacol peroxidation. In F Figureigure 88 itit is observable observable that that only only 1 1 min min after after starting starting the the reaction, reaction, there there is an is anincrease increase of the of absorbancethe absorbance around around the 470 the 470nm, nm,meaning meaning that thatsome some transformations transformations have have occurred. occurred. From From 1 min, 1 min, the absorbancethe absorbance at 47 at0 nm 470 increases nm increases significantly significantly and reaches and reaches its maximum its maximum value at value 15 min at 15which min means which thatmeans tetraguaiacol that tetraguaiacol (Figure (Figure 6 1a) concentration6(1a)) concentration achieves achieves its maximum. its maximum. Between 30 min and 48 h, the absorbance decreases, which could be explained either by the low stability of this product [37], or because, in the opinion of the authors, it could itself be a substrate to the enzyme. Despite that, this biotransformation reaction gave some important information: it proved that the reaction conditions chosen allow the enzymatic extract to work and to catalyze biotransformations, being effective in the transformation of simple phenols such as guaiacol.

Methods Protoc. 2020, 3, 25 10 of 12 Methods Protoc. 2020, 3, x FOR PEER REVIEW 10 of 12

Figure 8. Visible spectrophotometry of guaiacol oxidation products during reaction.

5. ConclusionsBetween 30 min and 48 h, the absorbance decreases, which could be explained either by the low stabilityIn conclusion, of this product the method[37], or because, described in is the a rapidopinion procedure of the authors, for the it preparation could itself of be a a ready-to-use substrate to peroxidase-richthe enzyme. Despite enzymatic that, this extract biotransformatio from turnip roots.n reaction It has gave the some advantage important of being information: versatile, sinceit proved this samethat the procedure reaction can conditions be employed chosen to obtain allow peroxidase the enzymatic from sources extract other to than work turnip, and toIn addition, catalyze therebiotransformations, is no need for furtherbeing effective purifications, in the which transformation turns the processof simple much phenols cheaper. such The as guaiacol. extract obtained was applied in biocatalysis and the results obtained indicate that turnip peroxidase extract is effective in5. Conclusions catalyzing the biotransformation of simple phenols, like guaiacol. The ability of the extract to polymerizeIn conclusion, simple phenolsthe method hints described for a possible is a rapid application procedure in bioremediation, for the preparation namely of ona ready the removal-to-use ofperoxidase phenol derivatives-rich enzymatic from extract wastewater. from turnip On the roots. other It hand,has the the advantage simplicity of being of this versatile, protocol since makes this it usefulsame procedure for didactic can purposes be employed in laboratory to obtain classes, peroxidase allowing from to demonstrate sources other how than to obtainturnip, an In enzymatic addition, extractthere is andno need the variousfor further factors purifications, on which which the activity turns the of an process enzyme much depends, cheaper. as The well extract as to verify obtained the actionwas applied of the in enzyme biocatalysis in reactions and the with results visual obtained effect. indicate that turnip peroxidase extract is effective in catalyzing the biotransformation of simple phenols, like guaiacol. The ability of the extract to Author Contributions: Conceptualization and methodology, A.M.L.S., M.d.C.B. and D.C.G.A.P.; formal analysis andpolymerize investigation, simple G.P.R.; phenols writing—original hints for a possible draft preparation, application G.P.R.; in bioremediation, writing—review namely and editing, on the removal A.M.L.S., M.d.C.B.of phenol and derivatives D.C.G.A.P.; from supervision, wastewater. A.M.L.S. On and the M.d.C.B. other hand, All authors the simpl haveicity read andof this agreed protocol to the publishedmakes it versionuseful of for the didactic manuscript. purposes in laboratory classes, allowing to demonstrate how to obtain an Funding:enzymaticThis extract research and was the funded various by FCT—Fundaçfactors on whichão para the a Ciactivityência e aof Tecnologia, an enzyme the depends, European Union,as well QREN, as to FEDER,verify the COMPETE, action of by the funding enzyme the cE3cin reactions centre (UIDB with/00329 visual/2020), effect. the LAQV-REQUIMTE (UIDB/50006/2020) and QOPNA (FCT UID/QUI/00062/2019) research units. Acknowledgments:Author Contributions:Thanks Conceptualization are due to the Universityand methodology, of Azores A.M.L.S., and University M.C.B. and of Aveiro. D.C.G.A.P.; formal analysis and investigation, G.P.R.; writing—original draft preparation, G.P.R.; writing—review and editing, A.M.L.S., Conflicts of Interest: The authors declare no conflict of interest and the funders had no role in the design of the M.C.B. and D.C.G.A.P.; supervision, A.M.L.S. and M.C.B. All authors have read and agreed to the published study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publishversion theof the results. manuscript. Funding: This research was funded by FCT – Fundação para a Ciência e a Tecnologia, the European Union, ReferencesQREN, FEDER, COMPETE, by funding the cE3c centre (UIDB/00329/2020), the LAQV-REQUIMTE (UIDB/50006/2020) and QOPNA (FCT UID/QUI/00062/2019) research units. 1. Dunn, P.J. The importance of green chemistry in process research and development. Chem. Soc. Rev. 2012, 41, Acknowledgments:1452–1461. [CrossRef Thanks][ PubMedare due to] the University of Azores and University of Aveiro. 2. Anastas, P.; Warner, J. Green Chemistry: Theory and Practice; Oxford University Press: New York, NY, USA, Conflicts of Interest: The authors declare no conflict of interest and the funders had no role in the design of the 1998; ISBN 9780198506980. study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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