Exploiting the Versatility of Aminated Supports ...

catalysts Article

Exploiting the Versatility of Aminated Supports Activated with Glutaraldehyde to Immobilize β-galactosidase from Aspergillus oryzae Hadjer Zaak 1,2,3 , Sara Peirce 1,4 , Tiago L. de Albuquerque 1,5 , Mohamed Sassi 3, * and Roberto Fernandez-Lafuente 1, * ID 1 2 3 4 5

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Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, 28049 Madrid, Spain; [email protected] (H.Z.); [email protected] (S.P.); [email protected] (T.L.d.A.) Food Biotechnology Division, Biotechnology Research Center (CRBt), Ali Mendjeli 91735, Algeria Nature and Life Science Faculty, Ibn Khaldoun University, Tiaret 14000, Algeria Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale—Università degli Studi di Napoli Federico II, P.le V. Tecchio 80, 80125 Napoli, Italy Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Fortaleza 60455-760, Brazil Correspondence: [email protected] (M.S.); [email protected] (R.F.-L.); Tel.: +213-795255505 (M.S.); +34-915-854-941 (R.F.-L.)

Received: 1 August 2017; Accepted: 18 August 2017; Published: 25 August 2017

Abstract: The enzyme β-galactosidase from Aspergillus oryzae has been immobilized in aminated (MANAE)-agarose beads via glutaraldehyde chemistry using different strategies. The immobilization on MANAE-supports was first assayed at different pH values (this gave different stabilities to the immobilized enzymes) and further modified with glutaraldehyde. Dramatic drops in activity were found, even using 0.1% (v/v) glutaraldehyde. The use of a support with lower activation permitted to get a final activity of 30%, but stability was almost identical to that of the just adsorbed enzyme. Next, the immobilization on pre-activated glutaraldehyde beads was assayed at pH 5, 7 and 9. At pH 7, full, rapid immobilization and a high expressed enzyme activity were accomplished. At pH 9, some decrease in enzyme activity was observed. Direct covalent immobilization of the enzyme was very slow; even reducing the volume of enzyme/support ratio, the yield was not complete after 24 h. The stability of the biocatalyst using pre-activated supports was about 4–6 folds more stable than that of the enzyme immobilized via ion exchange at pH 5, with small differences among them. Thus, the immobilization of the enzyme at pH 7 at low ionic strength on pre-activated glutaraldehyde supports seems to be the most adequate in terms of activity, stability and immobilization rate. Keywords: glutaraldehyde; enzyme immobilization; enzyme stabilization; enzyme inactivation

1. Introduction Immobilization of enzymes is usually required for their industrial implementation to solve the problem of enzyme solubility and the difficulties of enzyme reuse [1–4]. However, a proper immobilization protocol may also improve some enzyme features that may become a drawback in an industrial reactor, such as their moderate stability, inhibition by substrates or products, and moderate selectivity or specificity versus industrially relevant substrates (in many instances far from the physiological ones) [5–10]. Enzyme covalent immobilization may have some advantages compared to physical adsorption, such as prevention of enzyme desorption during operation and increased stability via multipoint covalent attachment [8,11]. However, it also has a problem: both enzyme and support must be discarded after enzyme inactivation [8]. Catalysts 2017, 7, 250; doi:10.3390/catal7090250

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The enzyme β-galactosidase (β-gal) from Aspergillus oryzae is a glycosylated enzyme whose structure has been recently resolved [12]. The pH/activity profile (with an optimal activity at acidic pH value) makes this enzyme not recommended to hydrolyze lactose in milk [13]. However, the enzyme is interesting for the hydrolysis of lactose in acid milk whey, and also for the production of galacto-oligosaccharides (prebiotics with great interest) or modification of galactose with other alcohols [14–18]. Among the strategies to covalently immobilize proteins, the use of glutaraldehyde may be of outstanding interest because of its high versatility [19–22] (Scheme 1), even though the exact glutaraldehyde chemistry is still under debate [23–25]. One accepted point is that glutaraldehyde forms some kind of cycles including the amino groups that make the amino-glutaraldehyde groups very stable [21,23,24]. This reagent is used to pre-activate supports having primary amino groups [24], and causes the support to become a heterofunctional one [26]. The activation under certain conditions permit to modify each primary amino group in the support with two molecules of glutaraldehyde, this amino-glutaraldehyde-glutaraldehyde group being very reactive with not ionized primary amino groups. Another alternative is the use of glutaraldehyde to crosslink enzyme and support after enzyme ion exchange immobilization on the aminated support [24]. In this strategy, both enzyme and support are modified under mild conditions to ensure that a maximum of one glutaraldehyde molecule react with each primary amino group. The high reactivity of the amino-glutaraldehyde moieties is introduced in both support and enzyme to get an intense multipoint covalent attachment, but also modifies the Lys groups not involved in the immobilization [24,27]. This may introduce a one point chemical modification of the primary amino groups of the enzyme (with positive or negative results on enzyme stability) or intra- or intermolecular crosslinking of the enzyme molecules, which should have a positive effect on enzyme stability [24]. The use of pre-activated support avoids this protein modification, but may have problems to give many enzyme support bonds, as amino-glutaraldehyde-glutaraldehyde groups are not very reactive with the ε-amino group of Lys (pK 10.7) at neutral pH value. This is a problem because the glutaraldehyde groups are not stable at alkaline pH values and therefore may not be used at these pH values [24]. In both cases, enzymes tend to become ionically immobilized before the enzyme reacts covalently with the support, because ion exchange is far more rapid than the covalent reaction [19,22,24]. Thus, permitting or not (e.g., using high ionic strength) the immobilization of β-gal via ion exchange on the support before the covalent immobilization, the results may become different [19,22]. Using this enzyme, the versatility using glutaraldehyde becomes even higher. β-gal may be immobilized via ion exchange at pH values ranging from 5 to 9 and this immobilization pH altered their final features, e.g., stability [28]. This was explained by the different orientation of the β-gal on the support surface. If this is the case, the pH of the first immobilization may also alter the number of enzyme-support linkages that may be achieved using glutaraldehyde, as the amount of nucleophiles may be different in different areas of the protein or the multipoint attachment may have different effects on enzyme stability depending on the protein area involved in the immobilization [29–32]. It should be considered that the most stable ionically immobilized enzyme was even less stable than the free enzyme under conditions where the enzyme had no tendency to aggregate, while, at pH 5 (near the ip of the protein), there is clear stabilization [28]. This glycosylated enzyme is difficult to be stabilized via multipoint immobilization because the sugar chains generate some hindrances to the enzyme/support reaction. Thus, the best results using this enzyme in terms of stabilization after immobilization have been reported using amino-epoxy supports, where a stabilization factor of 12 was obtained [33]. Now, we are going to try the immobilization of the enzyme on MANAE-agarose via glutaraldehyde chemistry using the different possibilities presented in this introduction. Agarose is a good support for enzyme immobilization, one of the reasons being that the only groups in the support are those that the researcher introduces, making it easier to understand the results [34].

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Glutaraldehyde

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Covalent enzyme/support bonds plus chemical modification Scheme 1. Schematic representation of the versatility of glutaraldehyde. Scheme 1. Schematic representation of the versatility of glutaraldehyde.

Thus, in this paper, we have intended the comparison of the different strategies that the Thus, of in glutaraldehyde this paper, we have intended the comparison of theenabled: differentthe strategies that the versatility versatility chemistry and aminated supports comparison between the of glutaraldehyde chemistry and aminated supports enabled: the comparison between the just ionically just ionically immobilized enzyme with the ionically immobilized plus glutaraldehyde treated immobilizedthe enzyme with the ionically immobilized plus glutaraldehyde treatedsupports, biocatalysts, biocatalysts, immobilization via ion exchange in glutaraldehyde pre-activated and the the immobilization via ion exchange in glutaraldehyde pre-activated supports, and the direct covalent direct covalent immobilization. All ion exchanges were performed at different pH values to permit immobilization. exchanges were performed at To different pH valuesthis to permit the adsorption of the adsorption ofAll theion enzyme via different areas [28]. our knowledge, is the first paper where the enzyme via different areas [28]. To our knowledge, this is the first paper where all these variables all these variables are considered in immobilization of any enzyme using glutaraldehyde chemistry. are considered in immobilization of any enzyme using glutaraldehyde chemistry. 2. Results and Discussion 2. Results and Discussion 2.1. 2.1. Immobilization Immobilization of of β-gal β-gal on on MANAE-Agarose MANAE-Agarose at at Different Different pH pH Values Values Figure It is rapid at all pHpH values; so Figure 11 shows shows the the immobilization immobilizationof ofβ-gal β-galatatpH pH5,5,7 7and and9.9. It very is very rapid at all values; rapid that, in the shortest time the first supernatant sample was taken, immobilization was almost so rapid that, in the shortest time the first supernatant sample was taken, immobilization was total, scarce among the threethe pHthree values. is thus even though thethough ionization almostwith total, withdifferences scarce differences among pHThis values. This is thus even the state of the support (pK of 6.8 and 9.8) and the enzyme will be very different at pH 5, 7 or 9. pH After ionization state of the support (pK of 6.8 and 9.8) and the enzyme will be very different at 5, immobilization, the activitythe recovered in all cases was almost 90% of90% theofoffered activity, in 7 or 9. After immobilization, activity recovered in all cases was almost the offered activity, agreement with thethe immobilization value in agreement with immobilizationcourses. courses.Figure Figure2 2shows showshow howthe the immobilization immobilization pH pH value affected the β-gal stability, showing the most and least stabilized enzymes at pH 5 and 9, affected the β-gal stability, showing the most and least stabilized enzymes at pH 5 and 9, respectively. respectively. This agrees with previous reports [28], although the new support is richer in amino This agrees with previous reports [28], although the new support is richer in amino groups. groups.

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Figure 1. Immobilization courses of β-gal on MANAE-agarose beads at: (A) pH 5; (B) pH 7; and

Figure 1. Immobilization courses of β-gal on MANAE-agarose beads at: (A) pH 5; (B) pH 7; and (C) (C) pH 9. Immobilization was performed at 25 ◦ C using 5 mM sodium acetate, sodium phosphate or pH 9. Immobilization performed at 25 °C using 5 Section. mM sodium acetate, sodium sodium bicarbonate, was respectively, as described in Methods A support having 60 µmolphosphate MANAE or sodium bicarbonate, respectively, as described in Methods Section. A support having groups/g of support was utilized. All experiments were performed in triplicate and the values60areμmol MANAE of support was utilized. All experiments were triplicate and the given groups/g as mean value ± the experimental error. Other specifications areperformed described inin Methods Section. Squares: Reference; Triangles: immobilization suspension; and Circles: immobilization supernatant. values are given as mean value ± the experimental error. Other specifications are described in Methods Section. Squares: Reference; Triangles: immobilization suspension; and Circles: immobilization supernatant.

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Time (hours) Figure 2. Effect of immobilization pH in the inactivation courses of the different β-gal-MANAE preparations. The inactivation was performed in 25 mM sodium phosphate at pH 7 and 50 ◦ C. The support presented 60 µmol MANAE groups/g of support. All experiments were performed Figure 2. Effect of immobilization pH in the inactivation of the different β-gal-MANAE in triplicate and the values are given as mean value ± the experimentalcourses error. Other specifications arepreparations. described in Methods Section. Squares: β-gal was immobilized at pH 5; phosphate Triangles: β-gal was The inactivation was performed in 25 mM sodium at pH 7 and 50 °C. The immobilized pH 7; and Circles: β-gal was immobilized at pH support at presented 60 μmol MANAE groups/g of 9.support. All experiments were performed in

triplicate and the values are given as mean value ± the experimental error. Other specifications are 2.2. Treatment of the MANAE-Agarose Immobilized Enzyme with Glutaraldehyde described in Methods Section. Squares: β-gal was immobilized at pH 5; Triangles: β-gal was This strategy is the easiest to transform the enzyme immobilizedatvia immobilized at pH 7; and Circles: β-gal was immobilized pHionic 9. exchange in a covalent immobilization. Using 1% (v/v) glutaraldehyde, the enzyme activity fully disappeared after a few minutes (results notofshown). The treatment ofImmobilized the immobilized enzyme with 0.1% (v/v) glutaraldehyde 2.2. Treatment the MANAE-Agarose Enzyme with Glutaraldehyde promoted the rapid inactivation of the enzymes immobilized at the three pH values (Figure 3A). This strategy is the easiest to transform thenow enzyme via ionic Although the glutaraldehyde chemical modification pH was 7 for all immobilized enzyme preparations, some exchange in a differences the inactivation courses be found depending on theenzyme immobilization values, covalentinimmobilization. Using could 1% (v/v) glutaraldehyde, the activitypH fully disappeared after reinforcing that each preparation presented a different stability. The enzyme immobilized at pH 9 lost a few minutes (results not shown). The treatment of the immobilized enzyme with 0.1% (v/v) theglutaraldehyde activity more rapidly than when enzyme was immobilized at pH 7 orimmobilized 5 (here, the inactivation promoted the the rapid inactivation of the enzymes at the three pH values was the slower). However, in the best case, only 10% of the initial activity was maintained. (Figure 3A). Although the glutaraldehyde chemical modification pH was now 7 for all enzyme The results could be derived from an intense enzyme-support reaction that leads to enzyme preparations, some differences in the inactivation courses could be found depending on the inactivation, or to a very negative effect of the glutaraldehyde modification in the enzyme properties. immobilization pH values, reinforcing that each preparation presented a different Thus, the effects of the modification with 0.1% (v/v) glutaraldehyde on the activity of the free enzyme stability. The enzyme immobilized pHThe 9 lost activity morepartially rapidlyinactivated than wheninthe have been checked (Figureat3B). freethe enzyme became theenzyme presencewas of immobilized glutaraldehyde (Figurethe 3B),inactivation but to a much lower (justHowever, around 10%) usingcase, the ionically at pH 7 or 5 (here, was theextent slower). in than the best only 10% of the initial absorbed enzyme. Even using 1% glutaraldehyde, a very high percentage of enzyme activity activity was maintained. was maintained. The results could be derived from an intense enzyme-support reaction that leads to enzyme Thus, it seems that the results were a consequence of the reaction of the enzyme and the support, inactivation, or to a very negative effect of the glutaraldehyde modification in the enzyme or of a more drastic modification of the immobilized enzyme by glutaraldehyde due to some distortion the effects the modification with 0.1% glutaraldehyde on the ofproperties. the ionically Thus, exchanged enzyme.ofTrying to avoid the activity losses,(v/v) we added 100 mM glucose to activity of the free enzyme checked 3B).the The free recovery enzyme(results became protect the active have center been [35], but this did(Figure not improve activity notpartially shown). inactivated in the

presence of glutaraldehyde (Figure 3B), but to a much lower extent (just around 10%) than using the ionically absorbed enzyme. Even using 1% glutaraldehyde, a very high percentage of enzyme activity was maintained.

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Figure 3. Effect on the enzyme activityof ofthe the incubation incubation of: or (B) freefree β-gal in thein the Figure 3. Effect on the enzyme activity of:(A) (A)β-gal-MANAE; β-gal-MANAE; or (B) β-gal presence of glutaraldehyde. The experiments were carried out in 25 mM sodium phosphate at pH 7 presence of◦ glutaraldehyde. The experiments were carried out in 25 mM sodium phosphate at pH 7 and 25 C. Other specifications are described in methods. A: (0.1% (v/v) glutaraldehyde). The support and 25 °C. Other specifications are described in methods. A: (0.1% (v/v) glutaraldehyde). The support had 60 µmol MANAE groups/g of support. All experiments were performed in triplicate and the had values 60 μmol groups/g All experiments were performed triplicate and areMANAE given as mean valueof ± support. the experimental error. (A) Squares: β-gal wasinimmobilized on the values are given as at mean ± theβ-gal experimental error.on (A) Squares: β-galatwas MANAE-agarose pH 5;value Triangles: was immobilized MANAE-agarose pH 7;immobilized and Circles: on MANAE-agarose at pH 5;onTriangles: β-gal was on1%MANAE-agarose at Circles: pH 7; and β-gal was immobilized MANAE-agarose at pHimmobilized 9. (B) Squares: glutaraldehyde; and 0.1%β-gal glutaraldehyde. Circles: was immobilized on MANAE-agarose at pH 9. (B) Squares: 1% glutaraldehyde; and Circles: 0.1% glutaraldehyde. As a last option, a support having a lower density of amino groups was prepared (having 60% Thus, it seems that themaximum results were a consequence of the reaction of ofthe enzyme and the of the amino groups of the activated support that has around 60 µmol MANAE groups per gram In this instance, theofionically exchanged enzyme was slower, support, or ofofa support). more drastic modification the immobilized enzyme inactivation by glutaraldehyde due to but still very significant; only 40% of the enzyme activity was maintained after 24 h of incubation some distortion of the ionically exchanged enzyme. Trying to avoid the activity losses, we added 100 To prevent inactivation, was the reduced withrecovery 1 mg/mL(results of mM (Figure glucose4A). to protect thefurther active enzyme center [35], but thisthe didbiocatalyst not improve activity sodium borohydride [36], which caused the final activity to be just around 30% of the initial one. not shown). Figure 4B shows that the stability of this preparation was very similar to that of the just adsorbed As a last option, a support having a lower density of amino groups was prepared (having 60% enzyme. In other words, this method did not seem to be very suitable to immobilize the β-gal.

of the amino groups of the maximum activated support that has around 60 μmol of MANAE groups per gram of support). In this instance, the ionically exchanged enzyme inactivation was slower, but still very significant; only 40% of the enzyme activity was maintained after 24 h of incubation (Figure 4A). To prevent further enzyme inactivation, the biocatalyst was reduced with 1 mg/mL of sodium borohydride [36], which caused the final activity to be just around 30% of the initial one. Figure 4B shows that the stability of this preparation was very similar to that of the just adsorbed enzyme. In other words, this method did not seem to be very suitable to immobilize the β-gal.

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Figure 4. Preparation and inactivation of β-gal-MANAE treated with 0.1% glutaraldehyde: (A) Activity Figure 4. Preparation and inactivation of β-gal-MANAE treated with 0.1% glutaraldehyde: (A) evolution of β-gal-MANAE immobilized at pH 5 and treated with 0.1% glutaraldehyde at pH 7 and Activity evolution of β-gal-MANAE immobilized at pH 5 and treated with 0.1% glutaraldehyde at 25 ◦ C using a support having 40 µmol MANAE groups/g of support. Other specifications are described pH 7 and 25 °C using a support having 40 μmol MANAE groups/g of support. Other specifications in methods. (B) Inactivation of different β-gal-MANAE preparations at pH 5 and 55 ◦ C. Circles: are described in methods. (B) Inactivation of different β-gal-MANAE preparations at pH 5 and 55 °C. β-gal-MANAE treated with glutaraldehyde in the presence of 100 mM glucose. All experiments were Circles: β-gal-MANAE treated with glutaraldehyde in the presence of 100 mM glucose. All performed in triplicate and the values are given as mean value ± the experimental error. Squares: experiments were performed in triplicate and the values are given as mean value ± the experimental β-gal-MANAE treated with glutaraldehyde in the absence of glucose. Triangles: β-gal-MANAE no error. Squares: β-gal-MANAE treated with glutaraldehyde in the absence of glucose. Triangles: treated with glutaraldehyde. β-gal-MANAE no treated with glutaraldehyde.

Therefore, rapid inactivation. inactivation. However, However, Therefore,ititseems seemsthat thatglutaraldehyde glutaraldehyde is is able able to to promote promote β-gal rapid glutaraldehyde and cannot cannot reach reach glutaraldehyde fixed fixed to to aa surface surface has has not not the the mobility mobility of free glutaraldehyde glutaraldehyde and internalpockets pockets of the way, it may be likely that that the enzyme may remain active using internal the enzyme. enzyme.That That way, it may be likely the enzyme may remain active glutaraldehyde pre-activate supports. using glutaraldehyde pre-activate supports. 2.3. 2.3.Immobilization Immobilizationofofβ-gal β-galon on Glutaraldehyde Glutaraldehyde Pre-Activated Pre-Activated Supports First, utilized in First,we wehave haveperformed performedthe theimmobilization immobilizationatatpH pH5,5,7 7and and9 9using usingthe thesame sameconditions conditions utilized the on MANAE-agarose via ion exchange, to permit this first stepthis in the immobilization. in immobilization the immobilization on MANAE-agarose via ion exchange, to permit first step in the Figure 5A shows Figure that in 5A thisshows case the enzyme situation was quite immobilization. that in thisremained case the active. enzymeHowever, remainedthe active. However, the different the quite different pH values. At pH 5, pH the values. enzymeAt was immobilized. At not this situationatwas different at the different pHalmost 5, the not enzyme was almost immobilized. At this pH, of theimmobilization main mechanism of immobilization should ion poor exchange due toofthe pH, the main mechanism should be ion exchange due be to the reactivity all poor reactivity of all amino groups of the enzyme. Furthermore, the presence of glutaraldehyde amino groups of the enzyme. Furthermore, the presence of glutaraldehyde dimers over the amino dimersseems over the amino the groups seems thetocapacity ofseveral the enzyme establish ionic groups to hinder capacity of to thehinder enzyme establish ionic to bridges withseveral the support bridges with thereadily support (the enzyme immobilized at this pH value onSteric nakedhindrances MANAE (the enzyme was immobilized atwas this readily pH value on naked MANAE supports). supports). Steric hindrances are reported as an important problem on the enzyme-support are reported as an important problem on the enzyme-support multi-interaction, due to the complexity

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multi-interaction, due to the complexity of both structures [26]. Thus, the enzyme remained fully active but mostly in the supernatant. At pH 7 and 9, a rapid immobilization took place, but, at pH 9, ofthe both structures [26]. Thus, the enzyme fully active but mostly in the At pH immobilization was accompanied by remained a certain enzyme inactivation, while, at supernatant. pH 7, the enzyme 7 and 9, a rapid immobilization took place, but, at pH 9, the immobilization was accompanied by remained almost fully active. a certain enzyme inactivation, while, at pH 7, the enzyme remained almost fully active.

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Figure5. 5. Effect Effect of the pH value value on Figure on the the immobilization immobilization courses courses ofof β-gal β-galononpre-activated pre-activated MANAE-glutaraldehyde supports. supports. The was performed at: (A) (B)5;pH or (C) MANAE-glutaraldehyde Theimmobilization immobilization was performed at: pH (A) 5; pH (B)7;pH 7; or pH 9 at 2525 °C◦using 5 mM sodium acetate, sodium phosphate or sodium bicarbonate, respectively, as (C) pH 9 at C using 5 mM sodium acetate, sodium phosphate or sodium bicarbonate, respectively, described in Methods Section. A support having 60 μmol MANAE groups/g of support was utilized. as described in Methods Section. A support having 60 µmol MANAE groups/g of support was utilized. experiments were performed in triplicate and the are values given value as mean ± the AllAll experiments were performed in triplicate and the values givenare as mean ± thevalue experimental experimental error. Other specifications are described in Methods Section. Triangles: immobilization error. Other specifications are described in Methods Section. Triangles: immobilization suspension; suspension; and Circles: immobilization of the immobilized enzymes and Circles: immobilization supernatant.supernatant. Incubation Incubation of the immobilized enzymes at pH 7 orat9pH after after 24 h in 500 mM sodiumproduced phosphatethe produced the of fullthe release of the enzyme 247horin9500 mM NaCl/25 mMNaCl/25 sodiummM phosphate full release enzyme immobilized

on MANAE-agarose [37,38], but all enzyme molecules remained immobilized on the glutaraldehyde preparations, showing the success of getting a covalent immobilization.

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immobilized on MANAE-agarose [37,38], but all enzyme molecules remained immobilized on the 9 of 14 glutaraldehyde preparations, showing the success of getting a covalent immobilization.

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Finally, Finally, we we tried tried to to get get the thedirect directcovalent covalentimmobilization immobilizationof ofthe theenzyme enzymeon onthe thesupport support[19,22]. [19,22]. In the immobilization immobilizationwill willproceed proceed most reactive primary amino group, andmain the In this this case, the byby thethe most reactive primary amino group, and the main effect of the pH is to control the protein reactivity, which should increase using a higher pH effect of the pH is to control the protein reactivity, which should increase using a higher pH value, value, pH8over this effect is detrimental forglutaraldehyde the glutaraldehyde groups stability Thus, to but at but pH at over this 8effect is detrimental for the groups stability [24].[24]. Thus, to this this we used andmM 250 sodium mM sodium phosphate. 6 shows that while the enzyme was goalgoal we used pH 8pH and8 250 phosphate. FigureFigure 6 shows that while the enzyme was almost almost not immobilized on support, MANAEsome support, some immobilization achieved using the not immobilized on MANAE immobilization was achievedwas using the glutaraldehyde glutaraldehyde MANAE support. However, the immobilization wasthan much slower when the was ion MANAE support. However, the immobilization was much slower when thethan ion exchange exchange was permitted and after 24 h no more than 20% of the enzyme was immobilized. A lower permitted and after 24 h no more than 20% of the enzyme was immobilized. A lower immobilization immobilization due to the reactivity of but glutaraldehyde, but in this case the rate is expectedrate dueistoexpected the low reactivity of low glutaraldehyde, in this case the difference became difference becameTovery significant. To improve the immobilization rate, we reduced the (increasing volume of very significant. improve the immobilization rate, we reduced the volume of enzyme enzyme (increasing the enzyme concentration to keepofthe total amount of protein) frompermitted 1:10 to 1:3. the enzyme concentration to keep the total amount protein) from 1:10 to 1:3. This to This permitted toof immobilize 80% of 48 thehenzyme immobilize 80% the enzyme after (Figure after 5B). 48 h (Figure 5B).

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Figure courses of β-gal on pre-activated MANAE-glutaraldehyde supports at pH at 8 Figure6.6.Immobilization Immobilization courses of β-gal on pre-activated MANAE-glutaraldehyde supports pH 8250 and 250sodium mM sodium phosphate. The immobilization was performed a: (A) or and mM phosphate. The immobilization was performed using a:using (A) 1/10; or1/10; (B) 1/3 (B) 1/3 support/enzyme suspension 25 ◦ C. A support having 60 µmolgroups/g MANAEofgroups/g support/enzyme suspension ratio at 25ratio °C. Aatsupport having 60 μmol MANAE support of support All experiments were in triplicate values are given was utilized.was All utilized. experiments were performed inperformed triplicate and the valuesand are the given as mean value as ± mean value ± the experimental error. Other conditions described in methods Section. Triangles: the experimental error. Other conditions are described in are methods Section. Triangles: immobilization immobilization and Circles: supernatant. immobilization supernatant. suspension; and suspension; Circles: immobilization

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2.4. Stability of the Different Preparations

Figure 7 shows the inactivation courses of the three glutaraldehyde preparations, compared to 2.4. of the Different Preparations thatStability of the enzyme just ionically adsorbed at pH 5. Both covalent enzyme preparations where the enzyme was previously ionically exchanged half-lives about six-fold higherto Figure 7 shows the inactivation courses ofpresented the three observed glutaraldehyde preparations, compared thanof that the enzyme just ionically adsorbed at 5. pHBoth 5, while the directly immobilized that theofenzyme just ionically adsorbed at pH covalent enzymecovalently preparations where the enzyme was the least stable one, showing a lower stability than even the just ionically immobilized enzyme was previously ionically exchanged presented observed half-lives about six-fold higher than enzyme. that of the enzyme just ionically adsorbed at pH 5, while the directly covalently immobilized enzyme Considering stability and immobilization rate, the it seems that immobilization at pH 7 was the least stableactivity, one, showing a lower stability than even just ionically immobilized enzyme. and Considering low ionic strength is the recommended method to immobilize β-gal using the glutaraldehyde activity, stability and immobilization rate, it seems that immobilization at pH 7 and chemistry. low ionic strength is the recommended method to immobilize β-gal using the glutaraldehyde chemistry. It should should be be remarked remarked that It that the the glutaraldehyde glutaraldehydebiocatalysts biocatalystsprepared preparedatatpH pH7 7and and9 9were werenow now almost identical in stability, while the ion exchange gave much lower stability for the enzyme almost identical in stability, while the ion exchange gave much lower stability for the enzyme immobilized at atpH pH9. 9. That That means means that thatthe thestabilization stabilizationachieved achievedwhen whenthe theenzyme enzymewas wasimmobilized immobilizedat immobilized at pH 9 by the glutaraldehyde reaction was higher that immobilizing the enzyme at pH However, pH 9 by the glutaraldehyde reaction was higher that immobilizing the enzyme at pH 7. 7. However, the the final stability fairly because similar the because thestability starting stability was the lower when the final stability becamebecame fairly similar starting was lower when immobilization immobilization was performed at pH 9 [28]. However, perhaps more covalent bonds could be was performed at pH 9 [28]. However, perhaps more covalent bonds could be formed when the formed when the enzyme was immobilized at pH 9. enzyme was immobilized at pH 9.

Residual activity, (%)

120 100 80 60 40 20 0 0

2

4

6

8

10

Tim e, (hours) Figure 7. Inactivation courses of different MANAE-glutaraldehyde-β-gal preparations at pH 5 and Figure 7. Inactivation courses of different MANAE-glutaraldehyde-β-gal preparations at pH 5 and 55 ◦ C. Experiments were performed as described in Methods Section; the enzymes were immobilized 55 °C. Experiments were performed as described in Methods Section; the enzymes were immobilized on a support having 60 µmol MANAE groups/g of support. All experiments were performed in on a support having 60 μmol MANAE groups/g of support. All experiments were performed in triplicate and the values are given as mean value ± the experimental error. Dashed line, Open circles: triplicate and the values are given as mean value ± the experimental error. Dashed line, Open circles: Free β-gal; Dashed line, Open Squares: β-gal-MANAE immobilized at pH 5; Open Triangles. Solid line: Free β-gal; Dashed line, Open Squares: β-gal-MANAE immobilized at pH 5; Open Triangles. Solid MANAE-glutaraldehyde-β-gal immobilized at pH 7 at low ionic strength; Open Circles, Solid line: line: MANAE-glutaraldehyde-β-gal immobilized at pH 7 at low ionic strength; Open Circles, Solid MANAE-glutaraldehyde-β-gal immobilized at pH 9 at low ionic strength; and Solid Squares, Solid line: MANAE-glutaraldehyde-β-gal immobilized at pH 9 at low ionic strength; and Solid Squares, line: MANAE-glutaraldehyde-β-gal immobilized at pH 8 at high ionic strength. Solid line: MANAE-glutaraldehyde-β-gal immobilized at pH 8 at high ionic strength.

3. 3. Materials Materials and and Methods Methods 3.1. Materials 3.1. Materials β-Galactosidase from A. oryzae (20 Units oNPG/mg of protein), o-nitrophenyl-β-galactopyranoside β-Galactosidase from A. oryzae (20 Units oNPG/mg of protein), (oNPG), glycidol, glutaraldehyde (25% (v/v), stabilized with methanol) sodium borohydride, o-nitrophenyl-β-galactopyranoside (oNPG), glycidol, glutaraldehyde (25% (v/v), stabilizedsodium with periodate ethylendiamine were sodium purchased from Sigma-Aldrich (St. Louis, were MO, USA). Four-percent methanol)and sodium borohydride, periodate and ethylendiamine purchased from CL agarose beads were from Healthcare. MANAE supports glyoxyl Sigma-Aldrich (St. Louis, MO, GE USA). Four-percent CL agarose beadswere wereprepared from GE from Healthcare. supports with awere modification the glyoxyl protocolsupports previously [40]; reaction was 24 h MANAE [39] supports prepared of from [39]described with a modification of time the protocol before reduction. All other reagents were of analytical grade. previously described [40]; reaction time was 24 h before reduction. All other reagents were of analytical grade.

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3.2. Preparation of Glutaraldehyde Agarose Beads Ten grams of MANAE agarose was suspended in 20 mL of 15% (v/v) glutaraldehyde in 200 mM phosphate buffer pH 7.0. The suspension was kept under mild stirring overnight at 4 ◦ C. After that, the support was filtered and washed with an excess of distilled water. The activated support was used immediately after preparation. This ensures that each primary amino in the support has been modified with two glutaraldehyde molecules [21,41]. 3.3. Standard Determination of Enzyme Activity This assay was performed by measuring the increase in absorbance at 380 nm produced by the release of o-nitrophenol in the hydrolysis of 10 mM oNPG in 100 mM sodium acetate at pH 4.5 and 25 ◦ C (ε was 10493 M−1 cm−1 under these conditions) [28]. This wavelength was chosen because this enzyme is active at acid pH values [12–14], and at these pH conditions, the ε of oNP is negligible at 410 nm. To start the reaction, 50–100 µL of the enzyme solution or suspension were added to 2.5 mL of substrate solution. One unit of activity (U) was defined as the amount of enzyme that hydrolyzes 1 µmoL of oNPG per minute under the conditions described previously. Protein concentration was determined using Bradford’s method [42]. 3.4. Immobilization of β-Galactosidase on Different Supports The immobilizations were carried out employing 20 oNPG units of beta-galactosidase per g of wet support (1 mg of enzyme per gram of support) and performed at 25 ◦ C. This low loading was used to prevent diffusion limitations that could distort the results. Thus, the commercial enzyme samples were dissolved in the corresponding volume of 5 mM sodium acetate at pH 5, sodium phosphate at pH 7 or sodium carbonate at pH 9. Then, ten grams of different supports (MANAE or glutaraldehyde agarose) were added. Reference suspensions were prepared in identical conditions, but using no activated agarose as matrix. In this matrix immobilization was negligible. An exception was made when ion exchange was not desired. In this case, 10 g of glutaraldehyde agarose was suspended in 30 mL of enzymatic solution prepared in 250 mM sodium phosphate at pH 8, maintaining the amount of enzyme per gram of support. The suspension was kept under stirring and samples of the suspension and the supernatant were withdrawn and enzyme activity was analyzed as described above after the desired immobilization time. When the different immobilizations were completed, the suspensions were washed, filtered and stored at 4 ◦ C for further characterization. In some instances, the enzyme immobilized on MANAE supports at the different pH values was washed and re-suspended, 5 mM sodium phosphate at pH 7 was used in both steps to ensure the pH of the final suspension. Then, 0.1% or 1% (v/v) glutaraldehyde was added to achieve some covalent enzyme-supports bonds [20]. After 1 h, the immobilized enzyme was washed and left at pH 7 for 24 h before storage it at 4 ◦ C. All experiments were performed in triplicate and the values are given as mean value ±the experimental error. 3.5. Thermal Stability of the Enzyme Preparations The enzyme was incubated at different pH values (5.0 and 7.0) in 25 mM of the sodium acetate or sodium phosphate respectively and a temperature that permitted a reasonable inactivation rate was selected. The objective was to compare the different immobilized enzyme stabilities under different conditions, in this case pH value and buffer nature. This may affect the ionization of the support/enzyme and that way, positive or negative interactions may be presented under certain pH values and not under other ones. At the desired times, samples of the inactivation suspension were withdrawn and the enzyme activity was measured using oNPG. All experiments were performed in triplicate and the values are given as mean ± the experimental error. Half-lives are given from the observed values from the inactivation courses, which follow a not first order inactivation showing some heterogeneity in the immobilized enzyme biocatalysts.

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4. Conclusions The versatility of glutaraldehyde has enabled to prepare β-gal biocatalysts with very good activity retention and a moderate stabilization. The glutaraldehyde molecules fixed on the support are unable to inactivate the enzyme. However, as described above, if free glutaraldehyde was added to the ionically exchanged enzyme, this became inactivated. Using free enzyme, this negative effect of glutaraldehyde modification is decreased. The effect was also decreased using a support with a lower activation degree. In any case, the stability of the enzyme did not improve and this immobilization strategy could be discarded. Considering activity, stability and immobilization rate, the proposed optimal protocol of β-gal immobilization from the seven studied possibilities is the immobilization on pre-activated supports at pH 7 and permitting a previous ion exchange of the enzyme on the support. The enzyme activity is almost fully preserved and a stabilization of six folds may be accomplished, in a very rapid and simple way. Acknowledgments: We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). HadjerZaak thanks the Algerian Ministry of higher education and scientific research for the fellowships. The pre-doctoral fellowships for de Albuquerque (CNPq, Brazil) and Miss Peirce (Universita’ degli Studi di Napoli Federico II) are also gratefully recognized. The help and suggestions of Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are gratefully recognized. Author Contributions: R.F.-L. and M.S. conceived and designed the experiments; R.F.-L. analyzed the data and wrote the paper; and H.Z., S.P., T.L.d.A. performed the experiments. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2.

3. 4. 5. 6. 7.

8.

9.

10.

11. 12.

Dicosimo, R.; McAuliffe, J.; Poulose, A.J.; Bohlmann, G. Industrial use of immobilized enzymes. Chem. Soc. Rev. 2013, 42, 6437–6474. [CrossRef] [PubMed] Cantone, S.; Ferrario, V.; Corici, L.; Ebert, C.; Fattor, D.; Spizzo, P.; Gardossi, L. Efficient immobilisation of industrial biocatalysts: Criteria and constraints for the selection of organic polymeric carriers and immobilisation methods. Chem. Soc. Rev. 2013, 42, 6262–6276. [CrossRef] [PubMed] Sheldon, R.A.; Van Pelt, S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. [CrossRef] [PubMed] Liese, A.; Hilterhaus, L. Evaluation of immobilized enzymes for industrial applications. Chem. Soc. Rev. 2013, 42, 6236–6249. [CrossRef] [PubMed] Rodrigues, R.C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernández-Lafuente, R. Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev. 2013, 42, 6290–6307. [CrossRef] [PubMed] Secundo, F. Conformational changes of enzymes upon immobilization. Chem. Soc. Rev. 2013, 42, 6250–6261. [CrossRef] [PubMed] Mateo, C.; Palomo, J.M.; Fernandez-Lorente, G.; Guisan, J.M.; Fernandez-Lafuente, R. Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol. 2007, 40, 1451–1463. [CrossRef] Garcia-Galan, C.; Berenguer-Murcia, A.; Fernandez-Lafuente, R.; Rodrigues, R.C. Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal. 2011, 353, 2885–2904. [CrossRef] Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Rodrigues, R.C.; Fernandez-Lafuente, R. Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts. Biotechnol. Adv. 2015, 33, 435–456. [CrossRef] [PubMed] Dos Santos, J.C.S.; Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R.C.; Fernandez-Lafuente, R. Importance of the Support Properties for Immobilization or Purification of Enzymes. ChemCatChem 2015, 7, 2413–2432. [CrossRef] Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme immobilization by adsorption: A review. Adsorption 2014, 20, 801–821. [CrossRef] Maksimainen, M.M.; Lampio, A.; Mertanen, M.; Turunen, O.; Rouvinen, J. The crystal structure of acidic β-galactosidase from Aspergillus oryzae. Int. J. Biol. Macromol. 2013, 60, 109–115. [CrossRef] [PubMed]

Catalysts 2017, 7, 250

13.

14.

15.

16.

17.

18. 19.

20.

21. 22.

23.

24.

25.

26.

27. 28.

29.

30.

13 of 14

Fischer, J.; Guidini, C.Z.; Santana, L.N.S.; de Resende, M.M.; Cardoso, V.L.; Ribeiro, E.J. Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae. J. Mol. Catal., B Enzym. 2013, 85–86, 178–186. [CrossRef] Urrutia, P.; Rodriguez-Colinas, B.; Fernandez-Arrojo, L.; Ballesteros, A.O.; Wilson, L.; Illanes, A.; Plou, F.J. Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae. J. Agric. Food Chem. 2013, 61, 1081–1087. [CrossRef] [PubMed] Vera, C.; Guerrero, C.; Illanes, A.; Conejeros, R. Fed-batch synthesis of galacto-oligosaccharides with Aspergillus oryzae β-galactosidase using optimal control strategy. Biotechnol. Prog. 2014, 30, 59–67. [CrossRef] [PubMed] Carevi´c, M.; Veliˇckovi´c, D.; Stojanovi´c, M.; Milosavi´c, N.; Rogniaux, H.; Ropartz, D.; Bezbradica, D. Insight in the regioselective enzymatic transgalactosylation of salicin catalyzed by β-galactosidase from Aspergillus oryzae. Process Biochem. 2015, 50, 782–788. [CrossRef] Porciúncula González, C.; Castilla, A.; Garófalo, L.; Soule, S.; Irazoqui, G.; Giacomini, C. Enzymatic synthesis of 2-aminoethyl β-D-galactopyranoside catalyzed by Aspergillus oryzae β-galactosidase. Carbohydr. Res. 2013, 368, 104–110. [CrossRef] [PubMed] Vera, C.; Guerrero, C.; Wilson, L.; Illanes, A. Synthesis of propyl-β-D-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae. Process Biochem. 2017, 53, 162–171. [CrossRef] Betancor, L.; López-Gallego, F.; Hidalgo, A.; Alonso-Morales, N.; Dellamora-Ortiz, G.; Mateo, C.; Fernández-Lafuente, R.; Guisán, J.M. Different mechanisms of protein immobilization on glutaraldehyde activated supports: Effect of support activation and immobilization conditions. Enzyme Microb. Technol. 2007, 39, 877–882. [CrossRef] López-Gallego, F.; Betancor, L.; Mateo, C.; Hidalgo, A.; Alonso-Morales, N.; Dellamora-Ortiz, G.; Guisán, J.M.; Fernández-Lafuente, R. Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports. J. Biotechnol. 2005, 119, 70–75. [CrossRef] [PubMed] Monsan, P. Optimization of glutaraldehyde activation of a support for enzyme immobilization. J. Mol. Catal. 1978, 3, 371–384. [CrossRef] Barbosa, O.; Torres, R.; Ortiz, C.; Fernandez-Lafuente, R. Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica. Process Biochem. 2012, 47, 1220–1227. [CrossRef] Migneault, I.; Dartiguenave, C.; Bertrand, M.J.; Waldron, K.C. Glutaraldehyde: Behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques 2004, 37, 790–802. [CrossRef] [PubMed] Barbosa, O.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Rodrigues, R.C.; Fernández-Lafuente, R. Glutaraldehyde in bio-catalysts design: A useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv. 2014, 4, 1583–1600. [CrossRef] Wine, Y.; Cohen-Hadar, N.; Freeman, A.; Frolow, F. Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis. Biotechnol. Bioeng 2007, 98, 711–718. [CrossRef] [PubMed] Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R.C.; Fernández-Lafuente, R. Heterofunctional supports in enzyme immobilization: From traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 2013, 14, 2433–2462. [CrossRef] [PubMed] Fernández-Lafuente, R.; Rosell, C.M.; Rodriguez, V.; Guisán, J.M. Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme Microb. Technol. 1995, 17, 517–523. [CrossRef] De Albuquerque, T.L.; Peirce, S.; Rueda, N.; Marzocchella, A.; Gonçalves, L.R.B.; Rocha, M.V.P.; Fernández-Lafuente, R. Ion exchange of β-galactosidase: The effect of the immobilization pH on enzyme stability. Process Biochem. 2016, 51, 875–880. [CrossRef] Grazú, V.; López-Gallego, F.; Montes, T.; Abian, O.; González, R.; Hermoso, J.A.; García, J.L.; Mateo, C.; Guisán, J.M. Promotion of multipoint covalent immobilization through different regions of genetically modified penicillin G acylase from E. coli. Process Biochem. 2010, 45, 390–398. [CrossRef] Mansfeld, J.; Vriend, G.; Van den Burg, B.; Eijsink, V.G.H.; Ulbrich-Hofmann, R. Probing the unfolding region in a thermolysin-like protease by site- specific immobilization. Biochemistry 1999, 38, 8240–8245. [CrossRef] [PubMed]

Catalysts 2017, 7, 250

31.

32. 33.

34. 35.

36.

37.

38.

39.

40.

41.

42.

14 of 14

Mansfeld, J.; Ulbrich-Hofmann, R. Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics. Biotechnol. Appl. Biochem. 2000, 32, 189–195. [CrossRef] [PubMed] Ulbrich-Hofmann, R.; Arnold, U.; Mansfeld, J. The concept of the unfolding region for approaching the mechanisms of enzyme stabilization. J. Mol. Catal. B Enzym. 1999, 7, 125–131. [CrossRef] Torres, R.; Mateo, C.; Fernández-Lorente, G.; Ortiz, C.; Fuentes, M.; Palomo, J.M.; Guisán, J.M.; Fernández-Lafuente, R. A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: Immobilization-stabilization of β-galactosidase from Aspergillus oryzae. Biotechnol. Prog. 2003, 19, 1056–1060. [CrossRef] [PubMed] Zucca, P.; Fernández-Lafuente, R.; Sanjust, E. Agarose and Its Derivatives as Supports for Enzyme Immobilization. Molecules 2016, 21, 1577. [CrossRef] [PubMed] Blanco, R.M.; Guisán, J.M. Protecting effect of competitive inhibitors during very intense insolubilized enzyme-activated support multipoint attachments: trypsin (amine)-agarose (aldehyde) system. Enzyme Microb. Technol. 1988, 10, 227–232. [CrossRef] Blanco, R.M.; Guisán, J.M. Stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives. Enzyme Microb. Technol. 1988, 11, 360–366. [CrossRef] Zaak, H.; Kornecki, J.F.; Siar, E.H.; Fernández-Lopez, L.; Corberán, V.C.; Sassi, M.; Fernández-Lafuente, R. Coimmobilization of enzymes in bilayers using PEI as a glue to reuse the most stable enzyme: Preventing PEI release during inactivated enzyme desorption. Process Biochem. 2017. [CrossRef] Peirce, S.; Virgen-Ortíz, J.J.; Tacias-Pascacio, V.G.; Rueda, N.; Bartolome-Cabrero, R.; Fernández-Lopez, L.; Russo, M.E.; Marzocchella, A.; Fernández-Lafuente, R. Development of simple protocols to solve the problems of enzyme coimmobilization. Application to coimmobilize a lipase and a β-galactosidase. RSC Adv. 2016, 6, 61707–61715. [CrossRef] Mateo, C.; Palomo, J.M.; Fuentes, M.; Betancor, L.; Grazú, V.; Lopez-Gallego, F.; Pessela, B.C.C.; Hidalgo, A.; Fernández-Lorente, G.; Fernández-Lafuente, R.; et al. Glyoxyl agarose: A fully inert and hydrophilic support for immobilization and high stabilization of proteins. Enzyme Microb. Technol. 2006, 39, 274–280. [CrossRef] Fernández-Lafuente, R.; Rosell, C.M.; Rodriguez, V.; Santana, C.; Soler, G.; Bastida, A.; Guisán, J.M. Preparation of activated supports containing low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method. Enzyme Microb. Technol 1993, 15, 546–550. [CrossRef] Corma, A.; Fornés, V.; Jordá, J.L.; Rey, F.; Fernandez-Lafuente, R.; Guisan, J.M.; Mateo, C. Electrostatic and covalent immobilisation of enzymes on ITQ-6 delaminated zeolitic materials. Chemical Communications 2001, 5, 419–420. [CrossRef] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).