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High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

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High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces catalysts Article High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces Alejandro H. Orrego 1, María Romero-Fernández 1, María del Carmen Millán-Linares 2 , Justo Pedroche 2, José M. Guisán 1,* and Javier Rocha-Martin 1,* 1 Department of Biocatalysis, Institute of Catalysis and Petrochemistry (ICP) CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain; [email protected] (A.H.O.); [email protected] (M.R.-F.) 2 Group of Plant Proteins, Instituto de la Grasa CSIC, Carretera Utrera Km 1, 41013 Seville, Spain; [email protected] (M.d.C.M.-L.); [email protected] (J.P.) * Correspondence: [email protected] (J.M.G.); [email protected] (J.R.-M.); Tel.: +34-91-585-4809 (J.M.G. & J.R.-M.) Received: 29 May 2020; Accepted: 15 June 2020; Published: 16 June 2020 Abstract: Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose. Keywords: protein stabilization; protein immobilization; 2-picoline borane; methacrylic support; microenvironment; biocatalysis; support hydrophilization 1. Introduction In recent years, the use of very rigid supports for enzyme immobilization has gained interest in the design of heterogeneous biocatalysts for chemical processes [1–4]. On the one hand, it allows the design of continuous flow enzymatic reactors with the corresponding intensification of bioprocesses [5,6]. On the other hand, the rigid supports and immobilized enzyme biocatalysts can be completely dried (e.g., with cold acetone) while keeping their porous structure intact [7,8]. Dry immobilized enzyme biocatalysts can be used in solvent-free reaction systems or completely anhydrous reaction media (organic solvents, supercritical fluids, etc.) [7,9,10]. The combination of the enzymatic catalysis, Catalysts 2020, 10, 676; doi:10.3390/catal10060676 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 676 2 of 10 enzyme stabilization, continuous flow reactors and solvent-free reactions is highly desirable for a more sustainableCatalysts 2019, chemical9, x FOR PEER industry REVIEW [6 ,11]. 2 of 10 An ideal rigid support must contain easily derivatizable reactive groups on its surface to enable the designAn ideal of dirigidfferent support immobilization must contain protocols easily derivatizable [4,12]. It is alsoreactive interesting groups thaton its these surface supports to enable are resistantthe design to mechanicalof different agitationimmobilization and diff erentprotocols reaction [4,12] conditions. It is also [2 inte]. Inresting this way, that simple these derivatization supports are processesresistant to of themechanical support surface,agitation enzyme and different immobilization reaction protocols conditions or additional[2]. In this modifications way, simple of immobilizedderivatization enzymes processes could of bethe designed. support Thesesurface immobilized, enzyme immobilization biocatalysts could protocols also be usedor additional in stirred tankmodifications reactors, whereof imm theyobilized might enzymes face several could challenges: be designed. the biphasic These reactionimmobilized systems, biocatalysts the need forcould the continuousalso be used control in stirred of pH, tank the reactors need for, where oxygen they bubbling, might face etc. several [13,14]. challenges: the biphasic reaction systems,The the highly need activatedfor the continuous methacrylate control carriers of pH, withthe need epoxy for oxygen groups bubbling, meet almost etc. [13, all14] essential. requirementsThe highly and activated are commercially methacrylate available carriers (Purolite with ECR epoxy from groups Purolite; meet Sepabeads almost and all Relizymes essential fromrequirements Resindion) and withare commercially different porosity available and (Purolite particle ECR size. from The Purolite; epoxy groups Sepabeads can beand hydrolyzed Relizymes withfrom acidResindion to yield) with diol different groups, porosity and these and can particle generate size. the The aldehyde epoxy groups groups can by be oxidation hydrolyzed with with the periodateacid to yield (Figure diol 1groups)[ 4]. Under, and these alkaline can generate conditions, the these aldehyde aldehyde-supports groups by oxidation promote with the the very periodate intense stabilization(Figure 1) [4] of. proteinsUnder byalkaline multipoint conditions, covalent these attachment, aldehyde orientating-supports the promote enzyme throughthe very its intense richest regionstabilization in lysine of residuesproteins [15by]. multipoint covalent attachment, orientating the enzyme through its richest region in lysine residues [15]. Figure 1. The functionalization of epoxy-activated supports based on methacrylate polymers with Figure 1. The functionalization of epoxy-activated supports based on methacrylate polymers with glyoxyl groups. glyoxyl groups. The hydrophilic/hydrophobic nature of methacrylate carriers is strongly determined by their polymericThe hydrophilic/hydrophobic composition. As a result, interactionsnature of methacrylate with polar or carriers hydrophobic is strongly regions determined of specific by enzyme their surfacespolymeric will composition be more or. lessAs favoureda result, [interact16]. Forions example, with polar highly or hydrophobic hydrophobic methacrylic regions of supports, specific suchenzyme as octadecyl-activatedsurfaces will be more ones, or less can favoured confer an [16] optimal. For example, hydrophobic highly microenvironment hydrophobic methacrylic to lipase activitysupports, and such stability as octadecy [3,9].l Unlike-activated other ones enzymes,, can confer the lipasean optimal surface hydrophobic is characterized microenvironment by significantly to extendedlipase activity lipophilic and regionsstability that [3, can9]. interactUnlike eotherffectively enzymes, with the the hydrophobic lipase surface supports is characterized [17]. In contrast, by mostsignificantly enzymes extended immobilized lipophilic in close regions contact that with can the interact hydrophobic effectively surfaces with can the undergo hydrophobic conformational supports changes[17]. In contrast, that seriously most aenzymesffect their immobilized activity and in stability close contact [18–20 ].with For the example, hydrophobic some hydrophobicsurfaces can pocketsundergo located conformational near the surfacechanges of that the seriously immobilized affect enzymes their activity can becomeand stability completely [18–20] exposed. For example, to the some hydrophobic pockets located near the surface of the immobilized enzymes can become medium due to the hydrophobic nature of the support. In general, enzymes are adversely affected by completely exposed to the medium due to the hydrophobic nature of the support. In general, the presence of hydrophobic surfaces [20–22]. enzymes are adversely affected by the presence of hydrophobic surfaces [20–22]. We envisioned a solution to this problem that consisted of the endpoint hydrophilization of the We envisioned a solution to this problem that consisted of the endpoint hydrophilization of the surface of these hydrophobic supports activated with aldehyde groups. This hydrophilization must surface of these hydrophobic supports activated with aldehyde groups. This hydrophilization must also include a large part of the surface of the support that is close to the surface of the enzyme. For also include a large part of the surface of the support that is close to the surface of the enzyme. For this purpose, we proposed the modification of the aldehyde groups that have not reacted with the this purpose, we proposed the modification of the aldehyde groups that have not reacted with the enzyme, with small hydrophilic amino ligands (e.g., glycine, lysine and aspartic) as blocking agents enzyme, with small hydrophilic amino ligands (e.g., glycine, lysine and aspartic) as blocking agents to generate microenvironments (cationic, anionic or neutral net charge) on the aldehyde-activated to generate microenvironments (cationic, anionic or neutral net charge)
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