Recent Trends in Biomaterials for Immobilization of Lipases for Application in Non-Conventional Media

catalysts

Review Recent Trends in Biomaterials for Immobilization of Lipases for Application in Non-Conventional Media

Robson Carlos Alnoch 1,2 , Leandro Alves dos Santos 3 , Janaina Marques de Almeida 2, Nadia Krieger 3 and Cesar Mateo 4,*

1 Departamento de Biologia, Faculdade de Filosoﬁa, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-900, São Paulo, Brazil; [email protected] 2 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19081, Centro Politécnico, Curitiba 81531-980, Paraná, Brazil; [email protected] 3 Departamento de Química, Universidade Federal do Paraná, Cx. P. 19061, Centro Politécnico, Curitiba 81531-980, Paraná, Brazil; [email protected] (L.A.d.S.); [email protected] (N.K.) 4 Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica (CSIC), Marie Curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain * Correspondence: [email protected]; Tel.: +34-915854768; Fax: +34-915854860

Received: 18 May 2020; Accepted: 18 June 2020; Published: 20 June 2020

Abstract: The utilization of biomaterials as novel carrier materials for lipase immobilization has been investigated by many research groups over recent years. Biomaterials such as agarose, starch, chitin, chitosan, cellulose, and their derivatives have been extensively studied since they are non-toxic materials, can be obtained from a wide range of sources and are easy to modify, due to the high variety of functional groups on their surfaces. However, although many lipases have been immobilized on biomaterials and have shown potential for application in biocatalysis, special features are required when the biocatalyst is used in non-conventional media, for example, in organic solvents, which are required for most reactions in organic synthesis. In this article, we discuss the use of biomaterials for lipase immobilization, highlighting recent developments in the synthesis and functionalization of biomaterials using different methods. Examples of effective strategies designed to result in improved activity and stability and drawbacks of the different immobilization protocols are discussed. Furthermore, the versatility of different biocatalysts for the production of compounds of interest in organic synthesis is also described.

Keywords: biomaterials; immobilization; lipases; biocatalysis; non-conventional media

1. Introduction Enzymes are proteins that catalyze reactions with high specificity and stereoselectivity and are widely used in industrial bioprocesses. The use of these biocatalysts has increased in recent years, mainly in the fields of organic synthesis [1,2], food modification, and biofuel development [3,4]. In organic synthesis, lipases are the most widely used class of enzymes [1]. In nature, these enzymes act on ester groups, but they are also able to catalyze hydrolysis or synthesis reactions with high chemo-, regio- and stereoselectivity with a wide variety of unnatural substrates [5], allowing their application to obtain different classes of organic compounds (Figure1). Currently, molecular biology and protein engineering strategies have been allied with reliable high-throughput-screening methods to obtain lipases with novel catalytic properties that have potential to compete with commercial lipases. Examples of recent successful approaches are the screening of novel enzymes from metagenomic libraries [6–11] and the combined protein engineering and enzyme immobilization (immobilized biocatalyst engineering) that was recently reviewed by Bernal et al. [12]. These strategies can be

Catalysts 2020, 10, 697; doi:10.3390/catal10060697 www.mdpi.com/journal/catalysts Catalysts 2020, 10, x FOR PEER REVIEW 2 of 29 Catalysts 2020, 10, 697 2 of 29 integrated to obtain immobilized lipases that have improved activity and stability in non- conventionalintegrated tomedia, obtain properties immobilized that lipases are required that have if improved lipases are activity to compete and stability with in chemical non-conventional routes commonlymedia, properties used in industry that are required[12]. Indeed, if lipases non-conv are toentional compete media, with chemicalespecially routes organic commonly media, are used commonlyin industry used [12 to]. Indeed,produce non-conventional manufactured products media, in especially biocatalysis organic [1]. media,In these are systems, commonly the water used to contentproduce is limited manufactured (i.e., theyproducts are aquo-restricted in biocatalysis media), [1]. which In these favors systems, synthesis the reactions water content and increases is limited the(i.e., productivity they are aquo-restricted of various media), processes, which especially favors synthesis those reactions involving and hydrophobic increases the productivitysubstrates. of Immobilizationvarious processes, has an especially important those role involving in the viability hydrophobic of these substrates. processes Immobilization since lipases do has not an perform important wellrole in insuch the systems viability in oftheir these free processes forms. since lipases do not perform well in such systems in their free forms.

FigureFigure 1. Examples 1. Examples of reactions of reactions catalyzed catalyzed by bylipases lipases with with different diﬀerent substrates substrates [13]. [13 ].

ResearchResearch into into new new materials materials has has contributed contributed in inrecent recent years years to tothe the development development of ofa multitude a multitude of ofnew new supports supports for for the the immobilization immobilization of oflipases, lipases, with with better better physical physical and and mechanical mechanical properties. properties. TheseThese new new materials materials include include magnetic magnetic nanoparticles nanoparticles [[14],14], carboncarbon nanotubes nanotubes [ 15[15]] synthetic synthetic polymers polymers [16 ], [16],mesoporous mesoporous and and electrospun electrospun materials materials [17], and[17], biomaterials. and biomaterials. They bringThey interestingbring interesting features, features, including includinggreater surfacegreater areas surface available areas for available immobilization, for immobilization, which allow an which effective allow enzyme an loadingeffective and enzyme improve loadingmass transferand improve during mass the reaction.transfer during the reaction. In Inthis this review, review, we we discuss discuss the the use use of ofbiomater biomaterialsials and and their their derivatives derivatives as assupports supports for for the the immobilizationimmobilization of of lipases asas e ffeffectiveective strategies strategies to improve to improve activity activity and stability. and stability. We review We experimental review experimentalstudies with studies a focus with on the a focus methods on the of immobilization. methods of immobilization. For each biomaterial For each (natural biomaterial polysaccharide), (natural polysaccharide),we briefly discuss we itsbriefly major propertiesdiscuss its before major turning properties to the before immobilization turning to protocols the immobilization used. However, protocolsbefore addressingused. However, the biomaterials, before addressing we briefly the biom describeaterials, the generalwe briefly features describe of lipases,the general since features they can of alipases,ffect the since selection they ofcan the affect support the andselection the method of the ofsupport immobilization. and the method We also of briefly immobilization. describe the We main alsomethods briefly useddescribe to immobilize the main methods lipases. used to immobilize lipases.

2. 2.Lipases: Lipases: General General Features Features and and Classical Classical Strategy Strategy of ofImmobilization Immobilization In Innature, nature, lipases lipases hydrolyze hydrolyze triacylglycerols, triacylglycerols, releasing releasing di- or di- monoacylglycerols, or monoacylglycerols, fatty acids, fatty and acids, glyceroland glycerol [18]. However, [18]. However, in practice, in practice, the definition the deﬁnition and andclassification classiﬁcation of lipases of lipases is issomewhat somewhat

Catalysts 2020, 10, 697 3 of 29 controversial, mainly due to the impossibility of strictly differentiating lipases from esterases. Traditionally, lipases (EC 3.1.1.3) have been defined as enzymes that hydrolyze long-chain triacylglycerols, which are water-insoluble substrates, whereas esterases (EC 3.1.1.1) hydrolyze small esters that are partially soluble in water, with both types of enzyme belonging to the sub-subclass of carboxylic ester hydrolases (EC 3.1.1) [3,5,18]. Later, Ali et al. [19] suggested classifying carboxylic ester hydrolases differently, dividing them into lipolytic esterases (EC 3.1 L) and non-lipolytic esterases (EC 3.1 NL). More recently still, Bracco et al. [20] suggested classifying lipases and esterases based on their ability to act in organic solvents that are immiscible in water (e.g., toluene), with low water activity (aw). In this review, we use the definition whereby lipases hydrolyze triacylglycerols or acyl esters with acyl chains of 10 carbons or more. Lipases have a common tertiary structure, the standard α/β hydrolase fold (Figure2A) [ 18]. In lipases, this structure has a central nucleus composed of parallel β-strands surrounded by α-helices and contains a catalytic triad composed of the amino acids Ser (which acts as a nucleophile during catalysis), His, and either Asp or Glu, with these amino acids being located at canonical positions in the α/β hydrolase fold [5,18]. Given that the natural substrates of lipases are hydrophobic, the active site usually has a hydrophobic nature. Typically, it also has a mobile subdomain called the lid. The lid can assume two conformations, closed and open. In the closed conformation, the lid blocks the active site, preventing entry of the substrate. In the open conformation, the substrate has free access to the active site (Figure2B). In aqueous solution, these two conformations are in equilibrium, with the closed conformation being favored. In the presence of hydrophobic substrates dispersed in water, the lipases are adsorbed at the hydrophobic interface and the equilibrium is shifted from the “closed” form of the enzyme to the “open” form, exposing the catalytic site, allowing substrate molecules to enter and suffer catalysis [5,18]. The conformational change of lipases at nonpolar-aqueous interfaces is known as interfacial activation and generally is associated with increased activity in the presence of hydrophobic interfaces [21,22]. However, although the presence of the lid is commonly correlated with the phenomenon of interfacial activation, lipases have been described that have a lid but do not present interfacial activation, for example, lipases from Burkholderia glumae and Pseudomonas aeruginosa [23,24]. Recently, Khan et al. [25] classified lipases into three different groups according to the structure of the lid domain: (1) lipases without a lid, (2) lipases with a lid formed by a single α-helix and (3) lipases with two or more α-helices forming the lid. The authors concluded that the lid domain also has a close relationship with the substrate specificity and thermostability of lipases [25]. Various strategies have been used over recent years to immobilize lipases: adsorption, the production of crosslinked enzyme crystals or aggregates, covalent binding, encapsulation and entrapment [26–29]. Among these strategies, physical adsorption is the most used, since, unlike other methods of immobilization, it does not cause large modifications in the native conformation of the enzyme or loss of catalytic activity [30]. Generally, the method consists of hydrophobic adsorption of the enzyme onto porous or non-porous supports and has been used as a standard technique in the purification, immobilization, and stabilization of lipases in a single-step process [21,22]. Physical adsorption onto hydrophobic supports fixes the lipase predominantly in its “open” form, producing more active and selective biocatalysts, especially when applied to the catalysis of complex reactions, such as kinetic resolution and regioselective hydrolyses or syntheses [31–33]. However, the hydrophobic part of lipases may favor the formation of dimers or aggregates with other hydrophobic proteins present in a crude extract, which may negatively affect immobilization [34,35]. In addition, the adsorption of lipases onto hydrophobic supports is reversible, meaning that the lipase can leach from the solid support, especially in the presence of detergents, additives or organic solvents [30], or even in the presence of the substrates and products of the reaction [36], which may limit the application of the biocatalyst. Lipase B from Candida antarctica (CALB) immobilized on polymeric beads (Lewatit VP OC 1600), named Novozym 435, is the commercially available immobilized lipase that is most used in biocatalysis, being applied in a wide variety of hydrolysis or synthesis reactions of interest to industry [1,37]. Catalysts 2020, 10, 697 4 of 29

However, Novozym 435 has various problems that may limit its industrial application, such as enzyme Catalystsleaching 2020 from, 10, x the FOR support, PEER REVIEW the fragility of the particles of the support under stirring, and dissolution4 of 29 of the support in polar solvents, like ethanol and methanol [37]. Therefore, it is necessary to search for dissolutionnew supports of the and support to develop in polar new solvents, immobilization like ethanol protocols. and methanol [37]. Therefore, it is necessary to search for new supports and to develop new immobilization protocols. (A)

(B)

FigureFigure 2. 2. (A(A) )Standard Standard αα/β/β hydrolasehydrolase fold fold of of the the superposed superposed structures structures of of the the lipases lipases from from BurkholderiaBurkholderia cepaciacepacia (blue)(blue) (PDB:1YS1) (PDB:1YS1) and and PseudomonasPseudomonas aeruginosa aeruginosa (gray)(gray) (PDB:1EX9). (PDB:1EX9). Zoom-in Zoom-in of of the the modeled modeled catalyticcatalytic site site and and the the calcium calcium ion ion (in (in red); red); ( (BB)) molecular molecular model model of of the the metagenomic metagenomic lipase lipase LipC12 LipC12 (PDB:6CL4)(PDB:6CL4) with with the the structure structure superposed superposed in in the the “open” “open” (red) (red) and and “closed” “closed” (blue) (blue) form. form.

InIn the the following following sections, sections, we we describe describe recent recent strategies strategies of ofimmobilization immobilization using using biomaterials biomaterials as supports,as supports, focusing focusing on strategies on strategies that improve that improve the acti thevity activity and stability and stability of the oflipases, the lipases, especially especially when theywhen are they used are in used reactions in reactions in organic in organic solvents. solvents. Hereafter, Hereafter, when when the theterm term “conversion” “conversion” appears, appears, it representsit represents the the number number of ofmols mols of ofester ester produced produced as as a apercentage percentage of of the the number number of of mols mols of of ester ester that that couldcould potentially potentially have have been been produced produced considering considering the the reagents. reagents.

3. Agarose

Agarose is a linear heteropolysaccharide composed of monomeric units of agarobiose (4-O-β-D- galactopyranosyl-3,6-anhydro-L-galactose) (Figure 3). It is the principal component of agar, a linear galactan extracted mainly from red seaweed (Phylum rhodophyta). Agarose has interesting features,

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3. Agarose Agarose is a linear heteropolysaccharide composed of monomeric units of agarobiose (4-O-β-d-galactopyranosyl-3,6-anhydro-l-galactose) (Figure3). It is the principal component of agar,Catalysts a linear 2020 galactan, 10, x FOR extracted PEER REVIEW mainly from red seaweed (Phylum rhodophyta). Agarose has interesting5 of 29 features, such as mechanical resistance, high hydrophilicity, pore and particle sizes that can be such as mechanical resistance, high hydrophilicity, pore and particle sizes that can be varied [38], and varied [38], and primary hydroxyl groups that can be chemically modified. These features have enabled primary hydroxyl groups that can be chemically modified. These features have enabled agarose to agarose to find many applications in molecular biology and biochemistry, where it is utilized as a matrix find many applications in molecular biology and biochemistry, where it is utilized as a matrix for for electrophoreticelectrophoretic analysis, purification, purification, and and immobili immobilizationzation of proteins of proteins [39]. The [39]. main The advantage main advantage of of usingusing agarose agarose as as a a matrix matrix forfor thethe immobilization of of enzymes enzymes is the is the presence presence of a oflarge a large number number of of hydroxylhydroxyl groups groups that allowthat allow crosslinking crosslinking with with different different agents, agents, as well as aswell functionalization as functionalization with with different groupsdifferent [39]. Forgroups example, [39]. For glyoxyl example, and glyoxyl epoxy and agarose epoxy agarose are often are used often to used immobilize to immobilize and stabilizeand enzymesstabilize via multipointenzymes via covalentmultipoint attachment. covalent attachment. In the case In ofthe lipases, case of octyl-agaroselipases, octyl-agarose (octyl-sepharose) (octyl- is thesepharose) most-used is commercialthe most-used support, commercial as it support, allows theas it lipase allows to the be lipase immobilized to be immobilized in the “open” in the form. However,“open” the form. adsorbed However, lipase the canadsorbed leach fromlipase thecan supportleach from in thethe presencesupport in of the high presence concentrations of high of concentrations of organic solvents or surfactants. This problem can be overcome by treatment with organic solvents or surfactants. This problem can be overcome by treatment with glutaraldehyde or glutaraldehyde or polymers like dextran or polyethyleneimine after immobilization, aiming to cross- polymers like dextran or polyethyleneimine after immobilization, aiming to cross-link the lipase to the link the lipase to the support [40–42]. However, this type of treatment can result in a loss of enzyme supportactivity [40– and42]. is However, costly. this type of treatment can result in a loss of enzyme activity and is costly.

FigureFigure 3. 3.Schematic Schematic representationrepresentation of of the the backbone backbone structure structure of agarose: of agarose: β-D-galactoseβ-d-galactose and 3,6- and 3,6-anhydro-anhydro-αα--lL-galactose, linked linked by by glycosidic glycosidic bonds bonds β-(1–4)β-(1–4) and and α-(1–3).α-(1–3). The Thelight light pink pinkcircles circles randomlyrandomly highlight highlight the the hydroxyl hydroxyl groups groups that that can can bebe chemically modified. modiﬁed.

AnotherAnother strategy strategy for for avoiding avoiding desorption desorption of the enzyme enzyme is isto touse use a heterofunctional a heterofunctional support. support. TheseThese supports supports are are produced produced through through thethe oxidationoxidation of of commercial commercial octyl-agarose octyl-agarose (glyoxyl-octyl (glyoxyl-octyl agarose) [43] or by bi-functionalization of the agarose (Figure 4A) with hydrophobic and aldehyde agarose) [43] or by bi-functionalization of the agarose (Figure4A) with hydrophobic and aldehyde groups (alkyl-aldehyde agarose) [44]. These heterofunctional supports allow the immobilization of groups (alkyl-aldehyde agarose) [44]. These heterofunctional supports allow the immobilization of lipases by adsorption onto a dense layer of hydrophobic moieties at neutral pH, followed by lipasesmultipoint by adsorption covalent onto attachment a dense layerbetween of hydrophobicthe adsorbed moietiesenzyme and at neutral the aldehyde pH, followed groups. The by multipoint main covalentadvantage attachment of the betweenalkyl-aldehyde the adsorbed support enzymeis that the and numbers the aldehyde of aldehyde groups. and alkyl The maingroups advantage on the of the alkyl-aldehydesurface of the support support can isbe that controlled. the numbers Furthermor of aldehydee, alkyl groups and with alkyl different groups chain on the sizes surface may be of the supportused can to allow be controlled. better control Furthermore, of the immobilization alkyl groups process, with producing diﬀerent biocatal chain sizesysts with may a bewider used range to allow betterof control properties of thethan immobilization are available with process, commercial producing supports. biocatalysts with a wider range of properties than are availableDifferent lipases with commercial have been immobilized supports. on glyoxyl-octyl and alkyl-aldehyde agarose supports; Ditheyﬀerent showed lipases improvements have been in immobilized activity (a phenomen on glyoxyl-octylon known as and hyperactivation), alkyl-aldehyde stability agarose at supports;high temperatures and high concentrations of organic solvents [43–50]. However, although these novel they showed improvements in activity (a phenomenon known as hyperactivation), stability at high strategies are useful for immobilization of lipases, they have some limitations: temperatures and high concentrations of organic solvents [43–50]. However, although these novel (i) alkaline pH values (around pH 10) are necessary to increase the reactivity of the nucleophilic strategiesresidues are exposed useful for at immobilizationthe protein surface, of lipases, especial theyly Lys have residues some limitations:(pKa 10.5). Some enzymes are (i)intrinsically alkaline pHunstable values under (around alkaline pH 10)conditions, are necessary for example, to increase the lipases the reactivity from Rhizomucor of the nucleophilic miehei residues(RML) exposed and Candida at the rugosa protein (CRL) surface, [46,51]; especially Lys residues (pKa 10.5). Some enzymes are intrinsically(ii) unstablethe additional under step alkaline of reduction conditions, that foris necessary example, to the establish lipases multiple from Rhizomucor covalent mieheilinkages(RML) and Candidabetween rugosathe support(CRL) and [46 the,51 enzyme]; may partially inactivate the enzyme; (ii) the(iii) additionalthe enzyme step must of have reduction at least thatone amino is necessary acid residue to establish that can multiplereact with covalent the aldehyde linkages betweengroups the of support the support and after the enzymethe adsorption. may partially inactivate the enzyme; Novel strategies have been proposed to improve these protocols, ranging from chemical (iii) the enzyme must have at least one amino acid residue that can react with the aldehyde groups modification using different agents by solid-phase chemistry [40,52] to the preparation of of theheterofunctional support after thesupports. adsorption. For example, the support octyl-glutamic agarose was proposed; it is obtained by derivatizing glyoxyl-octyl agarose with glutamic acid [48]. In this case, the

Catalysts 2020, 10, 697 6 of 29

Novel strategies have been proposed to improve these protocols, ranging from chemical modificationCatalysts 2020, using10, x FOR di PEERfferent REVIEW agents by solid-phase chemistry [40,52] to the preparation6 of 29 of heterofunctional supports. For example, the support octyl-glutamic agarose was proposed; it is obtainedimmobilization by derivatizing relies glyoxyl-octylon ionic interactions agarose withbetween glutamic the lipase acid [48and]. Inthe this support case, the after immobilization physical reliesadsorption on ionic interactionswith interfacial between activation, the lipase allowing and thestabilization support afterof the physical enzyme adsorption without incubation with interfacial at activation,alkaline allowingpH [48]. However, stabilization for the of theformation enzyme of withoutthese ionic incubation interactions, at alkalinethe protocol pH requires [48]. However, the for theimmobilized formation derivative of these to ionic be incubated interactions, at pH values the protocol around requires4.0 or 5.0, thewhich immobilized may lead to derivativecomplete to be incubatedloss of activity at pH of some values lipases around [48]. 4.0 or 5.0, which may lead to complete loss of activity of some Melo et al. [53] proposed a novel heterofunctional agarose support containing different groups lipases [48]. (hydrophobic or carboxylic/metal) for the adsorption of the enzyme via different regions, followed Melo et al. [53] proposed a novel heterofunctional agarose support containing different groups by covalent attachment with glutaraldehyde groups (Figure 4B). The main advantage of this support (hydrophobicis that it allows or carboxylic a single step/metal) immobilization for the adsorption at neutral of pH the and enzyme can, therefore, via diff erentbe used regions, with lipases followed by covalentthat are intrinsically attachment unstable with glutaraldehyde at alkaline pH. groupsMetagenomic (Figure lipase4B). TheLipC12 main and advantage CRL were ofsuccessfully this support is thatimmobilized it allows on a single this support, step immobilization presenting high at activity neutral and pH stability and can, after therefore, immobilization be used [53]. with lipases that are intrinsicallyThe use of lipases unstable immobilized at alkaline on pH.agarose Metagenomic supports in lipase organic LipC12 media and is limited, CRL were especially successfully in immobilizedsynthesis onreactions, this support, where the presenting highly hydrophilic high activity agarose and stability matrix retains after immobilization a significant amount [53]. of waterThe usethat of may lipases displace immobilized the equilibrium on agarose of the supports reaction. inThis organic can occur media even is limited,in functionalized especially in synthesisagaroses, reactions, since steric where hindrance the highly means hydrophilic that “inter agarosenal” hydroxyls matrix retainsreact poorly a significant with added amount reagents, of water thatmeaning may displace that it theis difficult equilibrium to achieve of the complete reaction. functionalization This can occur of eventhese ingroups functionalized [38]. In addition, agaroses, sincehydrophilic steric hindrance substrates means or products that “internal” of the reaction, hydroxyls such as react ethanol poorly and with glycerol, added may reagents, be retained meaning in the agarose matrix, which may lead to loss of enzyme activity or mass transfer problems. that it is difficult to achieve complete functionalization of these groups [38]. In addition, hydrophilic Indeed, there are relatively few reports of the use of lipases immobilized on agarose in pure substrates or products of the reaction, such as ethanol and glycerol, may be retained in the agarose organic solvents (100%) or solvent-free systems (i.e., systems in which the initial reaction medium matrix,contains which only may the lead substrates). to loss of Go enzymedoy [54] activity immobilized or mass lipases transfer from problems. Geobacillus thermocatenulatus, ThermomycesIndeed, there lanuginosus are relatively and Candida few reports antarctica of on the glyoxyl use of agarose lipases combined immobilized with a on new agarose amination in pure organicstrategy solvents that used (100%) different or solvent-free additives (dithiotreitol, systems (i.e., anthranilic systems acid, in whichmethyl theanthranilate, initial reaction and aniline), medium containsfollowed only by the drying substrates). with tert-butanol. Godoy [The54] dried immobilized biocatalysts lipases were from used Geobacillusin the ethanolysis thermocatenulatus of palm , Thermomycesolein in a lanuginosussolvent-free andsystem,Candida with antarcticathe maximumon glyoxyl conversion agarose obtained combined being 72% with in a 14 new h for amination the strategybiocatalyst that used prepared different with additivesT. lanuginosus (dithiotreitol, lipase. This anthranilic conversion acid,was 5-fold methyl higher anthranilate, than that obtained and aniline), followedfor the by non-dry drying withbiocatalysttert-butanol. (14% conversion The dried in biocatalysts 14 h), showing were usedthe positive in the ethanolysis effect of the of palmdrying olein in aprocedure. solvent-free system, with the maximum conversion obtained being 72% in 14 h for the biocatalyst preparedAlthough with T. few lanuginosus applicationslipase. in aquo-restricted This conversion media was have 5-fold been higherreported, than it is that important obtained to note for the non-drythat agarose-based biocatalyst (14% supports conversion have been in 14used h), as showing a proof of the concept positive of different effect of thestrategies, drying allowing procedure. an improvement of activity, stability, and selectivity of different lipases of interest in biocatalysis.

FigureFigure 4. Schematic 4. Schematic representation representation of of the the preparation preparation of of heterofunctional heterofunctional supports. (A (A) )Preparation Preparation of newof tailor-made new tailor-made alkyl-aldehyde alkyl-aldehyde supports; supports; (B) glutaraldehyde-activation (B) glutaraldehyde-activation of alkyl-aldehyde of alkyl-aldehyde support. support.

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Although few applications in aquo-restricted media have been reported, it is important to note that agarose-based supports have been used as a proof of concept of diﬀerent strategies, allowing an improvementCatalysts 2020, 10,of x FOR activity, PEER stability,REVIEW and selectivity of diﬀerent lipases of interest in biocatalysis. 7 of 29

4. Chitosan Chitosan-based materials have been used as matrices for the development of products in biomedicine and biotechnology. ChitosanChitosan isis a natural biopolymer derivedderived from chitin. It is non-toxic, bio-compatible, andand bio-degradable. Chitin is the second most abundant natural polysaccharide after cellulose and can be obtainedobtained from naturalnatural sources,sources, likelike exoskeletonsexoskeletons of arthropods,arthropods, such as insectsinsects and crustaceans [[55,56].55,56]. Chitosan isis composed composed of monomericof monomeric units units of β-(1,4)-2-amino-2-deoxy- of β-(1,4)-2-amino-2-deoxy-d-glucose,D-glucose, obtained obtained through thethrough partial the deacetylation partial deacetylation of chitin ( βof-(1,4)-2-acetamide-2-deoxy- chitin (β-(1,4)-2-acetamide-2-deoxy-d-glucose)D in-glucose) an alkaline in mediuman alkaline or throughmedium enzymaticor through hydrolysis enzymatic catalyzedhydrolysis by catalyzed chitin deacetylase by chitin deacetylase (Figure5)[ 55 (Figure,56]. The 5) [55,56]. great advantage The great ofadvantage immobilizing of immobilizing enzymes on enzymes chitosan ison the chitosan low cost is andthe thelow availability cost and the of diavailabilityfferent physical of different forms (powders,physical forms scales, (powders, hydrogels, scales, membranes, hydrogels, fibers, membra andnes, microspheres). fibers, and microsph It has reactiveeres). aminoIt has reactive (–NH2) andamino hydroxyl (–NH2) (–OH)and hydroxyl groups (–OH) that can groups be chemically that can be modified, chemically allowing modified, the introductionallowing the ofintroduction functional groupsof functional that can groups react withthat can amino react acid with residues amino at acid the residues protein surface. at the protein Furthermore, surface. the Furthermore, high density the of aminehigh density groups of in amine chitosan groups allows in chitosan the preparation allows the of reticulated preparation matrices, of reticulated in which matrices, the structural in which units the arestructural crosslinked units using are crosslinked reagents such using as epichlorohydrin,reagents such as glycidol, epichlorohydrin, polyaldehydes, glycidol, genipin, polyaldehydes, citric acid, sodiumgenipin, triphosphate citric acid, andsodium glutaraldehyde triphosphate [55 and–58]. glutaraldehyde Among these crosslinking [55–58]. Among agents, these genipin crosslinking (Figure6) hasagents, gained genipin prominence (Figure for6) has being gained less toxicprominence than glutaraldehyde for being less [ 59toxic], the than classical glutaraldehyde crosslinking [59], agent the forclassical chitosan crosslinking reticulation. agent for chitosan reticulation.

Figure 5. ChemicalChemical structure structure of ofchitosan. chitosan. Chitin Chitin can be can deacetylated be deacetylated by chitin by chitindeacetylases deacetylases or by ortreatment by treatment in hot alkaline in hot media. alkaline The media. backbone The of backbone chitosan is of a linear chitosan homopolymer is a linear of homopolymer β-(1,4)-2-amino- of β2-deoxy--(1,4)-2-amino-2-deoxy-D-glucose. The highlightedd-glucose. The groups highlighted (aqua = groups amino, (aqua light= pinkamino, = hydroxyl light pink and= hydroxyl mauve = and N- mauveacetyl) represent= N-acetyl) the represent sites that the can sites be chemically that can be modified. chemically modiﬁed.

Some importantimportant points points should should be be considered considered in the in usethe ofuse chitosan of chitosan for the for immobilization the immobilization of lipases: of lipases: (i) chitosan microspheres, beads, and ﬁbers show poor mechanical stability, which may result in (i) fragmentationchitosan microspheres, of these structures beads, and [60 fibers,61]. Reticulationshow poor mechanical of the chitosan stability, is important which may to produce result in a mechanicallyfragmentation resistant of these matrix structures and also[60,61]. toavoid Reticulation its dissolution of the chitosan under acidic is important conditions. to produce a (ii) dimechanicallyﬀerent agents resistant containing matrix reactive and also groups to av canoid beits useddissolution to control under solubility, acidic conditions. anionic properties, (ii) anddifferent hydrophobicity agents containing of chitosan. reactive One groups example can be is used glutaraldehyde; to control solubility, the bonds anionic formed properties, between and hydrophobicity of chitosan. One example is glutaraldehyde; the bonds formed between the polymer and this agent render the chitosan-matrix quite hydrophobic [62], which may be advantageous for lipase immobilization; (iii) immobilization may be affected by reactive groups in the chemical agent used to activate the chitosan, such as glutaraldehyde or ethylenediamine, and also by amino, hydroxyl and acetyl amine groups present in chitosan itself. Charged functional groups in the support may cause

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the polymer and this agent render the chitosan-matrix quite hydrophobic [62], which may be advantageous for lipase immobilization; (iii) immobilization may be affected by reactive groups in the chemical agent used to activate the chitosan, such as glutaraldehyde or ethylenediamine, and also by amino, hydroxyl and acetyl Catalystsamine 2020, groups10, x FORpresent PEER REVIEW in chitosan itself. Charged functional groups in the support may8 cause of 29 non-specific binding of the enzyme to the support, which can lead to loss of enzyme activity. Innon-specific some cases, binding it may of be the necessary enzyme to to block the support, the remaining which reactivecan lead groups to loss inof theenzyme support; activity. In (iv) similarly,some cases, reactive it may groups be necessary present to in block chitosan the mayremaining react with reactive substrates groups or in products the support; of the reaction, (iv) dependingsimilarly, reactive on the reactiongroups present conditions in chitosan used. For may example, react with the aminosubstrates groups or products may react of with the aldehydereaction, depending and ketone on groups, the reaction forming conditions Schiff bases used. that For aff ectexample, the solubility the amino of the groups polymer may react with aldehyde and ketone groups, forming Schiff bases that affect the solubility of the polymer

Figure 6. SchematicSchematic representation representation of the structure of ch chitosanitosan crosslinked with genipin according to Butler et al. [63]. [63].

Chitosan isis classicallyclassically activated activated by by derivatizing derivatizing the the amine amine groups groups with with glutaraldehyde glutaraldehyde (Figure (Figure7A). 7A).The exposedThe exposed reactive reactive group group of the of glutaraldehyde the glutaraldehyde then forms then covalentforms covalent bonds withbonds exposed with exposed amino, amino,thiol, phenolthiol, phenol or imidazole or imidazole groups groups of the of the enzyme, enzyme, conferring conferring high high stability stability againstagainst leaching. leaching. Glutaraldehyde-activated chitosan chitosan has been used toto immobilizeimmobilize severalseveral lipases.lipases. For For example, example, chitosan sequentiallysequentially activatedactivated with with glycidol, glycidol, ethylenediamine ethylenediamine (EDA), (EDA), and and glutaraldehyde glutaraldehyde (Figure (Figure7B) 7B)was was used used to immobilizeto immobilize CALB CALB (CALB-CH) (CALB-CH) [64 [64].]. CALB CALB was was also also immobilized immobilized onon glyoxylglyoxyl agarose

(CALB-GX) and octyl agarose (CALB-OC). CALB-CH was more stable at 65 ◦C and pH 7.0 (t 1 = 23½ min) (CALB-GX) and octyl agarose (CALB-OC). CALB-CH was more stable at 65 °C and pH 7.02 (t = 23 and in 70% dioxane (t 1 = 8 min)½ than was soluble CALB (t 1 = 3 min½ and t 1 = 1.5½ min, respectively). It was min) and in 70% dioxane2 (t = 8 min) than was soluble CALB2 (t = 3 min2 and t = 1.5 min, respectively). also much more stable in dioxane than were CALB-GX (t 1 = 0.28½ min) and CALB-OC (t 1 = 0.15½ min). It was also much more stable in dioxane than were CALB-GX2 (t = 0.28 min) and CALB-OC2 (t = 0.15 min).CALB-CH CALB-CH was used was toused synthesize to synthesize ethyl ethyl oleate oleate in near-critical in near-critical CO2 (100CO2 bar(100 and bar 29.9and ◦29.9C), with°C), with 46% 46%conversion conversion in 6.5 in h [6.564]. h However,[64]. However, the preparation the preparation lost more lost than more 90% than of its90% activity of its afteractivity four after cycles four of cyclesreuse [of64 ].reuse The [64]. authors The did authors not investigate did not investigate the effect ofthe supercritical effect of supercritical CO2 on the CO stability2 on the of stability CALB-CH of CALB-CHor soluble CALBor soluble in the CALB absence in the of theabsence reaction of the species, reacti noron species, the effects nor of the the effects conditions of the on conditions the integrity on theof the integrity support. of the support. In another study, chitosanchitosan waswas activatedactivated withwith ethylenediamineethylenediamine (EDA) and glutaraldehyde and used to immobilizeimmobilize thethe lipaselipase from fromCercospora Cercospora kikuchiim kikuchiim(a (a fungal fungal plant plant pathogen) pathogen) using using a fluidized-bed a fluidized- beddryer dryer system system [65]. [65]. The The immobilized immobilized preparation preparation had had a a high high lipase lipase activity activity andand lowlow water activity (aw == 0.08),0.08), suitable suitable features features for biodiesel for biodiesel synthesis. synthesis. It was Itused was in used the ethanolysis in the ethanolysis of coconut of oil coconut (Cocos nuciferaoil (Cocos oil). nucifera Conversionsoil). Conversions remained above remained 95% over above five 95% successive over five ethanolysis successive cycles. ethanolysis The fluidized- cycles. bedThe drying fluidized-bed was advantageous: drying was advantageous: lipases are generally lipases immobilized are generally in immobilized aqueous media in aqueous and sorbed media water and needssorbed to water be removed needs to before be removed use in organic before use media, in organic to prevent media, it from to prevent affecting it from the reaction affecting equilibrium the reaction orequilibrium interfering or interferingwith the withdiffusion the di ffofusion hydrophobi of hydrophobicc substrates. substrates. Other Other drying drying methods, methods, such such as lyophilization, maymay cause cause loss loss of of enzyme enzyme activity, activity, which which was notwas observed not observed using fluidized-bedusing fluidized-bed drying. drying. Another advantage of the fluidized-bed drying process was that immobilization and drying were achieved in a single step, which would be advantageous for industrial applications [65]. Recent works have used chitosan for the preparation of magnetic composite materials. Such materials are of great interest because magnetic separation is simple and more effective than filtration or centrifugation. The coating of magnetic nanoparticles with biopolymers like chitosan protects them against erosion and provides appropriate functional groups [14]. For example, magnetic Fe3O4 nanoparticles were prepared by the hydrothermal method and coated with chitosan activated with glutaraldehyde and then used to immobilize CRL [66]. The biocatalyst was used to hydrolyze castor

Catalysts 2020, 10, 697 9 of 29

Another advantage of the fluidized-bed drying process was that immobilization and drying were achieved in a single step, which would be advantageous for industrial applications [65]. Recent works have used chitosan for the preparation of magnetic composite materials. Such materials are of great interest because magnetic separation is simple and more effective than filtration or centrifugation. The coating of magnetic nanoparticles with biopolymers like chitosan protects them against erosion and provides appropriate functional groups [14]. For example, magnetic Fe3O4 nanoparticles were prepared by the hydrothermal method and coated with chitosan activated with glutaraldehyde and then used to immobilize CRL [66]. The biocatalyst was used to hydrolyze castor oil, with a degree of hydrolysis of 46% being obtained in 60 h at 34 ◦C. The activity was not stable: in the fourth hydrolysis cycle, the degree of hydrolysis was only 30%. The authors suggested that the loss of activity was due to “irreversible” adsorption of substrate and product on the support, since they washed the biocatalyst with tertiary butyl alcohol and PBS (phosphate-buffered saline 0.02 M, pH 7.0) to remove any substrate or product that had been retained on the support [66]. A similar strategy was used to prepare magnetic nanoparticles coated with chitosan; the preparation involved in situ crosslinking using citric acid, followed by activation with glutaraldehyde [67]. CALB was immobilized covalently on this support and the immobilized derivative was used to esterify itaconic anhydride with different polyols (Figure8). It was stable in the reaction medium (a mixture of toluene:Tetrahydrofuran (THF) (2:1)), retaining 80% of the initial activity after 5 reaction cycles, at 25 ◦C for 72 h [67]. The chitosan-coated nanoparticle matrix maintained its shape and its physicochemical properties to prevent lipase leaching. Therefore, the loss of activity appears to be due to denaturation of the enzyme itself, not decomposition of the support. In a similar manner, squaric acid (3,4-dihydroxy-3-cyclobutene-1,2-dione) was used as a crosslinking agent in the preparation of a co-polymer coat formed by collagen and chitosan [68]. The chitosan-collagen composite was used to coat magnetic Fe3O4 nanoparticles. The carboxylic groups of the CRL were firstly activated with (Dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) and then reacted with 1 NH2 groups of the coat. The activity of this immobilized preparation was 2-fold higher (52 U mg− ) 1 than that of nanoparticles prepared with traditional crosslinking agent glutaraldehyde (26 U mg− ). However, curiously, the amount of immobilized CRL was lower for nanoparticles prepared with squaric acid as a crosslinking agent. Nevertheless, both biocatalysts prepared with squaric acid and glutaraldehyde retained almost 70% of residual activity after 10 cycles of the hydrolysis of the olive oil at 37 ◦C, pH 7.4, for 30 min. Unfortunately, the ability of the biocatalysts to catalyze synthesis reactions in organic medium was not evaluated. Magnetic chitosan nanocomposites were also prepared using different functional ionic liquids. Several imidazole-based ionic liquids with different functional groups (carboxyl, alkyl, hydroxyl, and amino) were used to modify the chitosan [69]. The functionalized chitosan was used to encapsulate magnetic Fe3O4 nanoparticles. This support was used to immobilize porcine pancreatic lipase (PPL). In related works, mesoporous silica was functionalized using ionic liquids, resulting in a support with carboxyl-functionalized ionic liquids grafted onto the material [70,71]. These groups were then used to couple the mesoporous silica with chitosan, with the composite then being used to immobilize PPL. The immobilized PPL showed high hydrolytic activity in aqueous medium and better temperature stability than the free enzyme, although this depended on the functional group and the carbonic chain of the ionic liquid in the support. Unfortunately, the activity and stability of the biocatalysts in the organic media were not evaluated. New strategies for the functionalization of chitosan microspheres have been reported recently. For example, chitosan and chitin nanowhiskers were crosslinked using tannic acid [72]. This strategy resulted in a hybrid polymer composite with better mechanical strength than that of pure chitosan. The support was used to immobilize RML using activation with EDC and NHS. The biocatalyst was used in the synthesis of eugenyl benzoate from eugenol and benzoic acid. A conversion of 53% was obtained in 5 h at 50 ◦C. The reuse of the biocatalyst was not studied. Catalysts 2020, 10, 697 10 of 29 Catalysts 2020, 10, x FOR PEER REVIEW 10 of 29

Catalysts 2020, 10, x FOR PEER REVIEW 10 of 29

FigureFigureFigure 7. 7.ActivationActivation Activation of ofof chitosan chitosanchitosan using using thethe bifunctionalbibifunctionalfunctional reagentreagent glutaraldehyde.glutaraldehyde.glutaraldehyde. (A( (A)A ))Classical Classical Classical methodologymethodologymethodology for for for derivatizing derivatizing derivatizing the thethe primary primary aminoamino amino groupsgroups groups of of chitosan chitosan usingusing using glutaraldehydeglutaraldehyde glutaraldehyde as as a asa crosslinkinga crosslinkingcrosslinking agent. agent. ( B()B ()BPreparation )Preparation Preparation ofof of supportssupports supports basedbased based on on chitosan chitosan activatedactivated activated withwith with glycidol,glycidol, glycidol, ethylenediamine,ethylenediamine,ethylenediamine, and and and glutaraldehyde glutaraldehyde [64]. [ 64[64].].

FigureFigure 8. 8.Esteriﬁcation Esterification ofof itaconicitaconic anhydrideanhydride with polyols polyols catalyzed catalyzed by by CandidaCandida rugosa rugosa (CRL)(CRL) Figure 8. Esterification of itaconic anhydride with polyols catalyzed by Candida rugosa (CRL) immobilizedimmobilized on on magnetic magnetic chitosan chitosan nanoparticles nanoparticles [[65].65]. immobilized on magnetic chitosan nanoparticles [65]. Recently,Recently, CALB CALB was was physically physically encapsulatedencapsulated by oppositely charged charged polyions polyions in in a amixture mixture of of Recently, CALB was physically encapsulated by oppositely charged polyions in a mixture of sodiumsodium polyphosphate polyphosphate and and chitosanchitosan [73]. [73]. The The prepared prepared biocatalyst biocatalyst showed showed high high stability stability at 65 °C at 65(t½◦ C sodium polyphosphate and chitosan [73]. The prepared biocatalyst showed high stability at 65 °C (t½ = 366 min), at pH 4.0. It was applied in the synthesis of benzyl acetate from benzyl alcohol and vinyl = 366 min), at pH 4.0. It was applied in the synthesis of benzyl acetate from benzyl alcohol and vinyl

Catalysts 2020, 10, 697 11 of 29

Catalysts1 2020, 10, x FOR PEER REVIEW 11 of 29 (t 2 = 366 min), at pH 4.0. It was applied in the synthesis of benzyl acetate from benzyl alcohol acetate,and vinyl with acetate, a maximum with a conversion maximum of conversion 98% in 24 ofh at 98% 30 °C in [73]. 24 h Pinheiro at 30 ◦C[ et73 al.]. [74] Pinheiro prepared et al. a new [74] supportprepared for a newenzyme support immobilization, for enzyme divinyl immobilization, sulfone-activated divinyl sulfone-activated chitosan (Figure chitosan9). Divinyl (Figure sulfone9). (DVS)Divinyl is sulfone a versatile (DVS) agent is a versatile that can agent be used that canin bealkaline used inmedia, alkaline for media, example for exampleat pH 10.0, at pH while 10.0, glutaraldehyde,while glutaraldehyde, which is which the agent is the that agent is most that commonly is most commonly used to activate used to chitosan-based activate chitosan-based materials, ismaterials, unstable isunder unstable these under conditions. these conditions. Chitosan was Chitosan activated was with activated DVS at with pH DVS 10.0, at 12.5 pH and 10.0, 14.0. 12.5 DVS and activation14.0. DVS was activation an effective was an strategy eﬀective for strategy immobili forzation immobilization of CALB, which of CALB, showed which higher showed stability higher in differentstability inconditions diﬀerent of conditions pH and temperature of pH and than temperature did CALB than immobilized did CALB on immobilized chitosan activated on chitosan with glutaraldehydeactivated with glutaraldehyde [74]. Nevertheless, [74]. an Nevertheless, important draw an importantback of DVS drawback supports of is DVS that supportsthe lengthy is that spacer the armlengthy may spacer reduce arm the mayrigidity reduce induced the rigidity by the multipoint induced by covalent the multipoint attachment covalent [38,75] attachment Additionally, [38,75 it] isAdditionally, necessary to it block is necessary the remaining to block DVS the groups remaining of the DVS support groups after of immobilization the support after and immobilization this blocking stepand thiscan alter blocking the catalytic step can properties alter the catalytic of the lipase properties [38]. of the lipase [38].

Figure 9. Schematic representation ofof the the crosslinking crosslinking reaction reaction of of chitosan chitosan with with divinyl divinyl sulfone sulfone and and the thediﬀ erentdifferent functional functional groups groups onthe on activatedthe activated chitosan chitosan surface. surface.

The modificationmodification ofof chitosan chitosan with with hydrophobic hydrophobic agents agents changes changes its hydrophilicits hydrophilic character character to a moreto a morehydrophobic hydrophobic one. Some one. studiesSome havestudies explored have explored this type ofthis modification, type of modification, aiming to prepare aiming hydrophobic to prepare hydrophobicsupports that allowsupports the immobilizationthat allow theof immobilization lipases by adsorption. of lipases For example,by adsorption. hydrophobic For example, chitosan hydrophobicmicrospheres chitosan were prepared microspheres by reductive were prepared amination by (using reductive dodecyl amination aldehyde) (using for thedodecyl immobilization aldehyde) forofRML the immobilization [76]. The activity of ofRML the biocatalyst[76]. The activity was 3-fold of the higher biocatalyst than that was of 3-fold the free higher lipase. than It was that used of the to freeconvert lipase. sunflower It was used oil into to convert a structured sunflower lipid containingoil into a structured 49% of stearic lipid acid.containing The biocatalyst 49% of stearic was acid. used Thein successive biocatalyst cycles was of used transesterification, in successive maintainingcycles of transesterification, 70% of residual activity maintaining after the 70% tenth of cycleresidual [76]. activityLikewise, after Urrutia the tenth et al. cycle [77] modified[76]. Likewise, chitosan Urrutia microspheres et al. [77] with modified different chit alkylosan chainsmicrospheres (C4, C8, with and differentC12) (Figure alkyl 10 chains) and used (C4, themC8, and to immobilize C12) (Figure CALB 10) and RML.used them The hydrophobicity to immobilize CALB obtained and with RML. C8 Thewas greaterhydrophobicity than that obtained withwith C4,C8 butwas there greater was than no further that obtained increase inwith hydrophobicity C4, but there when was C12no furtherwas used. increase The biocatalysts in hydrophobicity were applied when in C12 the was selective used. hydrolysis The biocatalysts of fish were oil to applied obtain polyunsaturated in the selective hydrolysisfatty acids. of The fish length oil to of obtain the alkyl polyunsaturated chains linked to fa chitosantty acids. aff Theected length the activity, of the stability,alkyl chains and selectivitylinked to chitosanof the immobilized affected the enzyme activity, [77 stability,]. and selectivity of the immobilized enzyme [77]. As demonstrated, chitosan has been used successfully as a support for lipase immobilization in recentrecent years. The The different different strategies strategies for for the the functi functionalizationonalization of of this this polymer polymer provide provide a a wide wide variety variety of novel immobilization protocols. However, However, chitosan chitosan polymers polymers have have different different molecular molecular weights, weights, degrees of polymerization and and degrees of acetylation, depending mainly mainly on on the natural source and thethe way that the chitosan was obtained [78]. [78]. These These properties properties may may affect affect the characteristics of the matrix used in the immobilizationimmobilization processprocess and and also also the the properties properties of of the the biocatalyst, biocatalyst, which which may may lead lead to toproblems problems of of reproducibility. reproducibility.

Catalysts 2020, 10, 697 12 of 29 Catalysts 2020, 10, x FOR PEER REVIEW 12 of 29

Figure 10. SchematicSchematic representation representation of chitosan activated with different diﬀerent alkyl chains.

5. Cellulose Cellulose Cellulose is the most abundant renewable biopolymer on Earth. It is a linear polymer composed of dD-glucose-glucose residues residues linked linked by βby-(1,4) β-(1,4) glycosidic glycosidic bonds bonds (Figure (Figure 11). Some 11). advantages Some advantages in the utilization in the utilizationof cellulose of as cellulose a matrix as for a enzymematrix for immobilization enzyme immobilization are biocompatibility, are biocompatibility, high specific high surface specific area, surfaceexceptional area, mechanical exceptional properties, mechanical and properties, low cost [79and]. Celluloselow cost can[79]. beCellulose used in microcrystalline,can be used in microcrystalline,nanocrystalline, andnanocrystalline, chemically modified and chemically forms, ormodified even associated forms, or with even other associated materials, with such other as materials,nanocomposites such as or nanocomposit nanoparticleses [79 or,80 nanopart]. icles [79,80]. Despite the the great great potential potential of of ce cellulosellulose toto immobilize immobilize enzymes, enzymes, there there are arefew few reports reports of its of use its foruse immobilization for immobilization of lipases. of lipases. Karra-Châabouni Karra-Châ etabouni al. [81] et used al. [oxidiz81] useded cellulose oxidized fibers cellulose to immobilize fibers to Rhizopusimmobilize oryzaeRhizopus lipase oryzae (ROL)lipase by (ROL) physical by physical adsorption adsorption.. The Thecellulose cellulose fibers fibers (a (a commercial microcrystalline cellulose,cellulose, TECHNOCEL-150DM)TECHNOCEL-150DM) were were prepared prepared by by chemical chemical oxidation oxidation of of cellulose cellulose to togenerate generate carboxylic carboxylic groups groups on on the the cellulose cellulose surface surface (Figure (Figure 12 12A).A). TheThe contentcontent ofof carboxyliccarboxylic groups influencedinfluenced the adsorption of the lipase on the support, confirming confirming that the interactions of the enzyme with the cellulose were mainly electrostatic. The i immobilizedmmobilized derivative was used in the synthesis of butyl oleate in n-hexane, giving a conversion ofof 80% atat 3737 ◦°CC[ [81].81]. Koga et et al. al. [82] [82 ]functionalized functionalized cellulose cellulose paper paper with with 3-(trimethoxysilyl)propyl 3-(trimethoxysilyl)propyl methacrylate methacrylate and thenand thenimmobilized immobilized a lipase a lipase from fromBurkholderiaBurkholderia cepacia cepacia by hydrophobicby hydrophobic adsorption adsorption (Figure (Figure 12B). 12TheB). Theimmobilized immobilized derivative derivative was evaluated was evaluated in the in transesterification the transesterification of (R, ofS)-1-phenylethanol (R,S)-1-phenylethanol with vinyl with vinylacetate, acetate, using usingisopropyl isopropyl ether etheras the as so thelvent. solvent. The conversion The conversion to the to ester the ester(R)-1-phenylethylacetate (R)-1-phenylethylacetate was was50% 50%in 5 h, in at 5 h,23 at °C. 23 ◦C. Cellulose was modifiedmodified by using 3-aminopropyl-triethoxysilane3-aminopropyl-triethoxysilane (APTES) to introduce amino groups ontoonto thethe cellulosecellulose matrix matrix [ 83[83].]. Next, Next, glutaraldehyde glutaraldehyde was was used used to to introduce introduce aldehyde aldehyde groups groups for forthe the immobilization immobilization of CALB of CALB (Figure (Figure 13). 13). The The immobilized immobilized enzyme enzyme was usedwas used to esterify to esterify oleic oleic acid withacid withethanol ethanol in n-heptane, in n-heptane, giving giving 97% conversion 97% conversion in 1 h at in 60 1 ◦hC. at It 60 was °C. also It usedwas also in the used kinetic in the resolution kinetic resolutionof (R,S)-1-phenylethanol of (R,S)-1-phenylethanol in cyclohexane, in cyclohexane, with vinyl acetatewith vinyl as the acetate acyl donor. as the The acyl conversion donor. The of (conversionR)-1-phenylethanol of (R)-1-phenylethanol was 42% after was 5 h at42% 60 after◦C, with 5 h at an 60 enantiomeric °C, with an enantiomeric excess of product excess of of 99% product [83]. of 99%Cellulose [83]. can also be used to coat magnetic nanoparticles. Singh and Mukhopadhyay [84] coated Fe2OCellulose3 nanoparticles can also with be used cellulose to coat acetate magnetic and thennanoparticles. generated Singh aldehyde and groupsMukhopadhyay through oxidization[84] coated withFe2O3 HIO nanoparticles4. These aldehyde with cellulose groups acetate were then and usedthen generated to immobilize aldehyde CALB groups covalently. through The oxidization biocatalyst waswith appliedHIO4. These in the aldehyde synthesis groups of mono- were and then diglycerides used to immobilize from olive CALB oil. The covalently. best conversions The biocatalyst in 5 h werewas applied obtained in atthe 50 synthesis◦C, using oftert mono--butanol and asdiglycerid the solvent.es from In addition,olive oil. The the biocatalystbest conversions was used in 5 inh 11were successive obtained reaction at 50 °C, cycles, using giving tert-butanol a conversion as the to solvent. monoglycerides In addition, of 94% the ofbiocatalyst the theoretical was used maximum in 11 successive reaction cycles, giving a conversion to monoglycerides of 94% of the theoretical maximum (based on the starting substrate) [84], showing the high activity and stability of this preparation in tert-butanol.

Catalysts 2020, 10, 697 13 of 29

Catalysts 2020, 10, x FOR PEER REVIEW 13 of 29 (based on the starting substrate) [84], showing the high activity and stability of this preparation in tert-butanol. Catalysts 2020, 10, x HOFOR PEER βREVIEW HO 13 of 29 -(1-4) OH OH O HO O HO HO O O OH HO β O HO HO -(1-4) OOH HO OOH OOH HO OOH HO O On HO O OH HO OH O CelluloseHO OH O

OH OH n Figure 11. Schematic representation ofOH celluloseCellulose structure. The backbone isOH a linear homopolymer of

β-(1–4)-linked D-glucose residues. The light pink circles randomly highlight the hydroxyl groups that FigurecanFigure be chemically11. SchematicSchematic modified. representation representation of of cellulose cellulose structure. structure. The The backbone backbone is is a a linear linear homopolymer homopolymer of of ββ-(1–4)-linked-(1–4)-linked Dd-glucose-glucose residues. residues. The The light light pink pink circles circles ra randomlyndomly highlight highlight the the hydroxyl hydroxyl groups groups that that canNanocellulosecan be be chemically chemically has modified. modified. also been used to prepare nanocomposite conjugates with other polymers such as chitosan, with the intention of achieving a support with good mechanical stability. Elias et al. [85] preparedNanocelluloseNanocellulose beads hashas of alsoaalso nanocomposite beenbeen used used to to prepareof prepare chitosan nanocomposite nanocomposite and nanocellulose conjugates conjugates (denominated with with other other polymersCS-NC). polymers CRL such suchwasas chitosan, immobilized as chitosan, with with theon CS-NC intention the intention using of achieving glutaraldehyde of achieving a support a suas pportth withe crosslinking with good good mechanical agent.mechanical The stability. biocatalyst stability. Elias Elias was et al. etused [ 85al.] [85]inprepared the prepared synthesis beads beads of of a nanocompositeofbutyl a nanocomposite butyrate, ofwith chitosan of converchitosan andsions nanocelluloseand greaternanocellulose than (denominated 80% (denominated being CS-NC).obtained CS-NC). CRL in CRLfour was wassuccessiveimmobilized immobilized cycles on CS-NC onof reaction.CS-NC using using In glutaraldehyde addition, glutaraldehyde Raman as the spectroscopyas th crosslinkinge crosslinking and agent. optical agent. The Thefluorescence biocatalyst biocatalyst wasmicroscopy was used used in inshowedthe the synthesis synthesis the existence of butyl of butyl butyrate,of strong butyrate, withhydrogen conversionswith bondsconver greaterbetweensions greater than the 80% chitosan than being 80% and obtained being the nanocellulose. inobtained four successive in fourThe successivelipasecycles molecules of reaction. cycles wereof In reaction. addition, linked Into Raman addition,the surface spectroscopy Raman of the spectroscopy CS-NC and optical through and fluorescence imine optical bonds, fluorescence microscopy which were microscopy showed formed the showedthroughexistence theaof Schiff strongexistence base hydrogen mechanism of strong bonds hydrogen [85]. between bonds the chitosan between and the the chitosan nanocellulose. and the The nanocellulose. lipase molecules The lipasewere linkedmolecules to the were surface linked of theto the CS-NC surface through of the imineCS-NC bonds, through which imine were bonds, formed which through were formed a Schiff throughbase mechanism a Schiff base [85]. mechanism [85].

Figure 12.12.( A(A) Schematic) Schematic representation representation of the structureof the structure of carboxyl-cellulose. of carboxyl-cellulose. (B) Schematic (B representation) Schematic representationof the structure of of the cellulose structure modified of cellulose with 3-(trimethoxysilyl)propyl modified with 3-(trimethoxysilyl)propyl methacrylate. methacrylate. Figure 12. (A) Schematic representation of the structure of carboxyl-cellulose. (B) Schematic Magnetic cellulose nanocrystals (MCNCs) were prepared by a precipitation crosslinking method representationMagnetic cellulose of the nanocrystalsstructure of cellulose (MCNCs) modi werefied withprepared 3-(trimethoxysilyl)propyl by a precipitation crosslinkingmethacrylate. method and used to immobilize Pseudomonas cepacia lipase (PCL) [80]. The biocatalyst was applied in the and used to immobilize Pseudomonas cepacia lipase (PCL) [80]. The biocatalyst was applied in the asymmetric hydrolysis of (R,S)-ketoprofen ethyl ester, where the yield of (R)-ketoprofen was 43%, asymmetricMagnetic hydrolysis cellulose nanocrystalsof (R,S)-ketoprofen (MCNCs) ethyl were ester, prepared where by the a precipitationyield of (R)-ketoprofen crosslinking was method 43%, with an enantiomeric excess of product of 85%. In addition, the biocatalyst was used in six successive andwith used an enantiomeric to immobilize excess Pseudomonas of product cepacia of 85%. lipase In addition, (PCL) [80].the biocatalyst The biocatalyst was used was in applied six successive in the reaction cycles, retaining over 66% of its initial activity in the last cycle [80]. asymmetricreaction cycles, hydrolysis retaining of over (R,S 66%)-ketoprofen of its initial ethyl activity ester, inwhere the last the cycle yield [80]. of ( R)-ketoprofen was 43%, Bacterial cellulose is an alternative source of cellulose. At the most basic level, the chemical with Bacterialan enantiomeric cellulose excess is an of alternative product of source85%. In of addition, cellulose. the At biocatalyst the most was basic used level, in sixthe successive chemical composition of bacterial cellulose is identical to that of plant cellulose, with an ordered crystalline reactioncomposition cycles, of retainingbacterial celluloseover 66% isof identicalits initial toactivity that of in plant the last cellulose, cycle [80]. with an ordered crystalline structure of high purity. Recent works have explored the use of bacterial cellulose for immobilization structureBacterial of high cellulose purity. is Recent an alternative works have source explor ofed cellulose. the use of At bacterial the most cellulose basic level, for immobilization the chemical of lipases by physical adsorption and covalent binding [79,86,87]. Interesting properties were reported compositionof lipases by of physical bacterial adsorptioncellulose is andidentical covalent to that binding of plant [79,86,87]. cellulose, Interestingwith an ordered properties crystalline were for the biocatalysts in these works, such as high activity and better stability than the free enzyme at structurereported offor high the biocatalystspurity. Recent in worksthese works,have explor suched as the high use activity of bacterial and bettercellulose stability for immobilization than the free pH and temperature values typically used in biocatalytic processes. However, the properties of the ofenzyme lipases at bypH physical and temperature adsorption values and covalent typically binding used in [79,86,87]. biocatalytic Interesting processes. properties However, were the biocatalysts were not evaluated in organic media. reportedproperties for of thethe biocatalystsbiocatalysts inwere these not works, evaluated such in as organic high activity media. and better stability than the free Semi-synthetic polymers, such as hydroxypropyl methylcellulose (HMC), have also been used to enzymeSemi-synthetic at pH and polymers, temperature such values as hydroxypropyl typically used methylcellulose in biocatalytic (HMC), processes. have alsoHowever, been used the immobilize lipases. Badgujar et al. [88] immobilized four commercial lipases in HMC by entrapment. propertiesto immobilize of the lipases. biocatalysts Badgujar were notet al.evaluated [88] immobilized in organic media.four commercial lipases in HMC by entrapment.Semi-synthetic The best polymers, results were such obtainedas hydroxypropyl using the methylcellulose immobilized Pseudomonas (HMC), have fluorescens also been lipase, used towith immobilize yields of over lipases. 90% forBadgujar the synthesis et al. of [88] 21 different immobilized β-amino four ester commercial compounds, lipases via the in aza-Michael HMC by entrapment. The best results were obtained using the immobilized Pseudomonas fluorescens lipase, with yields of over 90% for the synthesis of 21 different β-amino ester compounds, via the aza-Michael

Catalysts 2020, 10, 697 14 of 29

Catalysts 2020, 10, x FOR PEER REVIEW 14 of 29 The best results were obtained using the immobilized Pseudomonas fluorescens lipase, with yields of overaddition 90% reaction, for the synthesis with remarkable of 21 di ffchemoselectivierent β-aminoty ester (>94%) compounds, in the carbon-nitrogen via the aza-Michael bond formation addition reaction,[88]. with remarkable chemoselectivity (>94%) in the carbon-nitrogen bond formation [88]. CelluloseCellulose is aa viableviable alternativealternative forfor immobilizingimmobilizing enzymesenzymes andand variousvarious formsforms ofof cellulosecellulose havehave beenbeen usedused toto immobilizeimmobilize lipases:lipases: cellulose fibers,fibers, cellulose paper, cellulosecellulose acetate,acetate, nanocellulose, hydroxypropylhydroxypropyl methylcellulosemethylcellulose andand bacterialbacterial cellulose.cellulose. In addition,addition, functionalizationfunctionalization strategiesstrategies thatthat areare usedused forfor otherother supportssupports cancan bebe applied,applied, includingincluding oxidationoxidation toto generategenerate carboxyliccarboxylic groupsgroups andand additionaddition of methacryloxy,methacryloxy, amino,amino, epoxyepoxy andand aldehydealdehyde groups.groups. CelluloseCellulose has also beenbeen usedused toto formform compositescomposites withwith otherother materialsmaterials suchsuch asas chitosanchitosan andand magneticmagnetic nanoparticles.nanoparticles.

FigureFigure 13.13. Schematic representation of the structure of cellulosecellulose functionalizedfunctionalized with-aminopropyl- triethoxysilanetriethoxysilane (APTES)(APTES) andand glutaraldehyde.glutaraldehyde.

VariousVarious methods methods have have been been used used to immobilize to immobilize lipases lipaseson cellulosic on cellulosicsupports, including supports, includingelectrostatic electrostatic adsorption, adsorption, hydrophobic hydrophobic adsorption, adsorption,covalent bonding, covalent entrapment bonding, and entrapment precipitation- and precipitation-cross-linking,cross-linking, giving immobilization giving immobilization efficiencies from effi ciencies10.5% to from 95%. 10.5%The highest to 95%. immobilization The highest immobilizationefficiency (95%) e wasfficiency achieved (95%) usin wasg achievedcellulose usingpaper cellulosefunctionalized paper with functionalized methacryloxy with groups, methacryloxy which groups,increase whichin the increase hydrophobicity in the hydrophobicity of the support of the and support favorand interaction favor interaction with lipases with lipases[82]. Good [82]. Goodimmobilization immobilization efficiencies effi cienciesfor lipases for have lipases also been have achieved also been with achievedhydroxypropyl with methylcellulose hydroxypropyl methylcellulosevia entrapment via(>91%), entrapment using (a> protocol91%), using without a protocol washing without or decanting washing or [88], decanting and with [88], cellulose- and with cellulose-acetate-coatedacetate-coated Fe2O3 nanoparticles Fe2O3 nanoparticles via covalentvia bonding covalent (85%) bonding [84]. In (85%)the latter [84 case,]. In the the surface latter layer case, theof the surface support layer has of hydrophobic the support hasgroups hydrophobic that may groupshave enabled that may greater have interaction enabled greater of the interactionenzyme with of thethe enzymesupport with[84]. theIt should support be [84 noted]. It should that immobi be notedlization that immobilization efficiency is not effi theciency only is notcriterion the only for criterionevaluating for the evaluating effectiveness the eff ectivenessof a support. of a support. For example, For example, CALB CALBwas covalently was covalently immobilized immobilized on oncellulose cellulose functionalized functionalized with with amino amino groups, groups, with with an an immobilization immobilization efficiency efficiency of of only only 10.5% 10.5% [83]. [83]. However,However, comparedcompared toto NovozymeNovozyme 435435 (CALB(CALB immobilizedimmobilized onon anan acrylicacrylic resin),resin), thethe esterificationesterification activityactivity for synthesissynthesis of ethylethyl oleateoleate waswas similar,similar, andand thethe kinetickinetic resolutionresolution ofof ((RR,,SS)-)-αα-phenylethanol-phenylethanol waswas better.better. RelativelyRelatively few reuse cycles have been used with lipases immobilized on cellulosic supports. AfterAfter 33 cycles,cycles, RhizopusRhizopus oryzaeoryzae lipaselipase immobilizedimmobilized onon modifiedmodified cellulosecellulose fibersfibers gavegave onlyonly 25%25% ofof thethe initial esterification activity [81]. After 8 cycles, CRL immobilized on a nanocomposite of chitosan and nanocellulose had lost around 50% of its initial activity in the synthesis of butyl butyrate [85].

Catalysts 2020, 10, 697 15 of 29

Catalystsinitial esteriﬁcation2020, 10, x FOR activityPEER REVIEW [81]. After 8 cycles, CRL immobilized on a nanocomposite of chitosan15 of and 29 nanocellulose had lost around 50% of its initial activity in the synthesis of butyl butyrate [85]. Finally, Finally,after 11 glycerolysisafter 11 glycerolysis cycles, Candida cycles, antarctica Candida lipaseantarctica immobilized lipase immobilized onto cellulose onto acetate-coated cellulose acetate- Fe2O3 coatednanoparticles Fe2O3 nanoparticles retained 94% retained of its initial 94% activity of its initial [84]. activity [84].

6.6. Starch Starch StarchStarch is aa naturalnatural polymer polymer composed composed of amylose,of amylose, a linear a linear polysaccharide polysaccharide containing containingα-(1,4)-linked α-(1,4)- linkedanhydroglucose anhydroglucose units (AGU), units and(AGU), amylopectin, and amylopectin, which has αwhich-(1,4)-AGU has α chains-(1,4)-AGU joined chains at branch-points joined at branch-pointsby α-(1,6)-linkages by α-(1,6)-linkages (Figure 14)[89 (Figure]. 14) [89].

FigureFigure 14. Schematic representationrepresentation of of the the structure structure of starch. of starch. Two Two different different polysaccharides polysaccharides are present are presentin starch: in ( Astarch:) linear (A (1,4)-linked) linear (1,4)-linkedα-d-glucose α- (amylose)D-glucose and(amylose) (B) highly and branched (B) highlyα-d branched-glucose (amylopectin). α-D-glucose (amylopectin).The light pink circlesThe light randomly pink circle highlights randomly the hydroxyl highlight groups the hydroxyl that can groups be chemically that can modified. be chemically modified. Starch films have many advantages, such as low cost, renewability, biodegradability, and ease of production,Starch films and havehave been many used advantages, to immobilize such severalas low lipasescost, renewability, [90]. The matrix biodegradability, of the starch geland allows ease ofthe production, entrapment and of thehave enzyme been used and, afterto immobilize gel drying, several the resulting lipases biocatalyst[90]. The matrix can be of used the instarch different gel allowsreaction the media. entrapment For example, of the theenzyme lipase and, from afterBurkholderia gel drying, cepacia the(BCL) resulting was immobilizedbiocatalyst can by entrapmentbe used in differentin a starch reaction film, and media. successfully For example, applied the in lipase the synthesis from Burkholderia of aroma esterscepacia (1-octyl (BCL) was acetate immobilized and 1-pentyl by entrapmentoctanoate) in in media a starch containing film, and ionic successfully liquids mixed applied with in organic the synthesis solvents of [aroma91]. After esters optimization (1-octyl acetate of the andreaction 1-pentyl conditions, octanoate) the conversion in media containing was greater ionic than liquids 99% after mixed 24 h atwi 25th ◦organicC for two solvents different [91]. reactions After optimizationin different media: of the (i)reaction the synthesis conditions, of 1-octyl the conver acetatesion using was neatgreater methyl than tert-butyl 99% after ether 24 h (MTBE)at 25 °C andfor twoalso different using diethyl reactions ether in/[BMIM][PF different media:6] (9:1), (i) and the (ii) sy thenthesis synthesis of 1-octyl of 1-pentyl acetate octanoateusing neat using methyl diethyl tert- butylether /ether[BMIM][Cl] (MTBE) (9:1) and [also89]. using Corn starchdiethyl films ether/[BMIM][PF were also prepared6] (9:1), and (ii) used the to synthesis immobilize of 1-pentyl CRL by octanoateentrapment using [92]. diethyl The biocatalyst ether/[BMIM][Cl] was applied (9:1) in [89]. the enantioselective Corn starch films resolution were also of (preparedR,S)-menthol and usingused tovinyl immobilize acetate asCRL the by acyl entrapment donor (Figure [92]. The 15), biocatal with tolueneyst was as applied the solvent in the atenan 35◦tioselectiveC. The conversion resolution of of(R ()-mentholR,S)-menthol to (R using)-menthyl vinyl acetateacetate esteras the was acyl 33%, donor with (Figure an enantiomeric 15), with toluene excess as of the product solvent of at greater 35°C. Thethan conversion 99% after 48of h(R [92)-menthol]. to (R)-menthyl acetate ester was 33%, with an enantiomeric excess of product of greater than 99% after 48 h [92]. Some structural properties of starch can be modified to meet specific requirements [93]. One example is oxidation with sodium periodate in an acidic medium, which leads to the formation of a

Catalysts 2020, 10, 697 16 of 29

Some structural properties of starch can be modiﬁed to meet speciﬁc requirements [93]. Catalysts 2020, 10, x FOR PEER REVIEW 16 of 29 OneCatalysts example 2020, 10 is, x oxidationFOR PEER REVIEW with sodium periodate in an acidic medium, which leads to the formation16 of 29 of a polyaldehyde chain (Figure 16)[94]. The aldehyde groups make this derivative suitable for polyaldehyde chain (Figure 16) [94]. The aldehyde groupsgroups makemake thisthis derivativederivative suitablesuitable forfor immobilization by multipoint covalent attachment with amino groups of the enzyme. immobilization by multipoint covalent attachment with amino groups of the enzyme.

Figure 15.15. Kinetic resolution of ((R,,S)-menthol)-menthol withwith vinylvinyl acetateacetate catalyzedcatalyzed byby CRLCRL immobilizedimmobilizedimmobilized inin starchstarch ﬁlmsfilms byby entrapment.entrapment.

Figure 16. Schematic representation of the chemical structure ofof polyaldehydepolyaldehyde starch.starch..

This strategystrategy waswas usedused toto immobilizeimmobilize aa lipaselipase fromfrom RhizopusRhizopus sp.sp.sp. ononon magneticmagneticmagnetic nanoparticlesnanoparticlesnanoparticles coatedcoated withwith twotwo didifferentfferent crosslinkerscrosslinkers (i)(i) polyaldehydepolyaldehyde starchstarch (MDSNIL)(MDSNIL) andand (ii)(ii) glutaraldehydeglutaraldehyde (MNGCL)(MNGCL) [95]. [95]. The The immobilized immobilized enzyme enzyme tolerated tolerated moderately moderately acidic acidic environments, environments, pH pH 6.0–6.5. 6.0–6.5. At AtpH pH 9.0, 9.0, MNGCL MNGCL maintained maintained 53% 53% of of initial initial activity, activity, while while MDSNIL MDSNIL maintained maintained 65%, 65%, indicating aa higher tolerancetolerance of of the the lipase lipase in these in these preparations preparat thanions is obtainedthan is obtained with the glutaraldehydewith the glutaraldehyde crosslinked lipase.crosslinked The biocatalyst lipase. The showed biocatalyst stability showed at 60 stability◦C, with at relative 60 °C, activities with relative of 55% activities and 60% of being 55% obtained,and 60% afterbeing 2 obtained, h of incubation, after 2 h for of MNGCLincubation, and for MDSNIL, MNGCL respectively. and MDSNIL, The respectively. residual activity The residual of MDSNIL activity in nof-hexane MDSNIL was in 52.1% n-hexane after was incubation 52.1% after for 2 h.incubation The reuse for experiments 2 h. The reuse showed experiments that MNGCL showed retained that 46.8%MNGCL residual retained activity 46.8% after residual 6 reuses, activity while after MDSNIL 6 reuses, retained while 53.6% MDSNIL of initial retained lipolytic 53.6% activity of underinitial thelipolytic same activity conditions. under Together the same with conditions. the great Together advantage with of the the immobilized great advantage biocatalyst of the immobilized being easily separatedbiocatalyst from being the easily reaction separated medium from through the reaction the application medium of through an external the application magnetic field, of an the external results ofmagnetic the work field, highlight the results the potential of the work application highlight of the polyaldehyde potential application starch as the of crosslinkingpolyaldehyde agent. starch as the crosslinking agent. 7. Alginate 7. Alginate 7. AlginateAlginate is an anionic linear polysaccharide present in the cell walls of brown algae, including speciesAlginate of Laminaria is an anionicand Ascophyllum linear polysaccharide. Alginates arepresent composed in the ofcell di wallsfferent of amounts brown algae, of (1–4) including linked βspecies-d-mannuronic of Laminaria acid andand and Ascophyllumα-l-guluronic.. AlginatesAlginates acid units areare (Figure composedcomposed 17)[ ofof96 differentdifferent]. They are amountsamounts low-cost, ofof non-toxic,(1–4)(1–4) linkedlinked and β- D-mannuronic acid and α-L-guluronic acid units (Figure 17) [96]. They are low-cost, non-toxic, and biocompatible. Alginates are soluble but, for enzyme immobilization, they are co-precipitated with the enzyme using cations that act as ionic bridges between different molecules of alginate. The most commonly used cation is Ca2+ [97].

Catalysts 2020, 10, x FOR PEER REVIEW 17 of 29

Figure 17. Schematic representation of the chemical structure of sodium alginate. The highlighted Catalysts 2020, 10, 697 17 of 29 groups (aqua = carboxyl and light pink = hydroxyl) represent the sites that can be chemically modified. biocompatible. Alginates are soluble but, for enzyme immobilization, they are co-precipitated with As is the case with starch, alginate in gel form is used to immobilize lipases by entrapment or the enzyme using cations that act as ionic bridges between different molecules of alginate. The most encapsulation. For example, a lipase from Aspergillus niger was entrapped in calcium alginate beads commonly used cation is Ca2+ [97]. Catalystsand used 2020 in, 10 the, x FORchemo-enzymatic PEER REVIEW epoxidation of α-pinene (Figure 18) in ethyl acetate as the solvent17 of 29 [96]. The conversion of α-pinene to α-pinene oxide was 65% in 24 h. The biocatalyst was used in 10 successive reactions, with a loss of activity of 36% after the tenth reaction [98]. The pores of the reticulated alginate network are large enough that small molecular weight molecules (<20 kDa) can diffuse into and out of the alginate beads [99]. A disadvantage of this support is that small proteins may leach into the bulk medium. For example, a crude extract containing lipases of Yarrowia lipolytica was immobilized by microencapsulation in alginate [100]. The immobilization itself was successful, with the immobilized biocatalyst initially having high hydrolytic activity against p-nitrophenyl laurate; however, 50% of the activity had leached out of the alginate beads by the end of the reaction [100]. The optimization of immobilization parameters can reduce the leaching of small proteins, for example, by controlling the amount of alginate and the concentration of Ca2+ to fine tuneFigure the 17. gel Schematic properties representation [101]. of the chemical structurestructure of sodiumsodium alginate.alginate. The highlighted groupsAnother (aqua(aqua strategy= =carboxyl carboxyl to avoid and and light leaching light pink pink= ishydroxyl) =to hydrouse the representxyl) alginate represent the sitesin the combination that sites can that be chemically can with be other chemically modified. polymers, supportsmodified. or nanoparticles. For example, the lipase from oleaginous seeds of Pachira aquatica was immobilizedAs is the by case entrapment with starch, using alginate a mixture in gel of formalginate is used and polyvinyl to immobilize alcohol lipases (PVA). by The entrapment thermal orstability encapsulation.As is increased, the case Forwithwith example, starch,the lipase alginate a lipasekeeping in from gel 60% formAspergillus of its is originalused niger to immobilizeactivitywas entrapped after lipases 4 h at in 50by calcium °C,entrapment whereas alginate noor encapsulation.beadsactivity and was used detected For in ex theample, after chemo-enzymatic incubation a lipase from of the epoxidation Aspergillus free enzyme niger of α under -pinenewas entrapped these (Figure conditions. in18 )calcium in ethyl Nevertheless, alginate acetate beads as this the andsolventprocedure used [96 in ].did the The not chemo-enzymatic conversion prevent the of αloss-pinene epoxidation of activity to α-pinene of thα-pinenee oxidebiocatalyst, was(Figure 65% given 18) in 24in that h.ethyl Theits acetateactivity biocatalyst as decreased the was solvent used by [96].in50% 10 Theafter successive conversion five successive reactions, of α -pinenecycles with aof lossto hydrolysis α-pinene of activity oxideof ofp-nitrophenyl 36% was after 65% thein palmitate 24 tenth h. The reaction [102]. biocatalyst [98]. was used in 10 successive reactions, with a loss of activity of 36% after the tenth reaction [98]. The pores of the reticulated alginate network are large enough that small molecular weight molecules (<20 kDa) can diffuse into and out of the alginate beads [99]. A disadvantage of this support is that small proteins may leach into the bulk medium. For example, a crude extract containing lipases of Yarrowia lipolytica was immobilized by microencapsulation in alginate [100]. The immobilization itself was successful, with the immobilized biocatalyst initially having high hydrolytic activity against p-nitrophenyl laurate; however, 50% of the activity had leached out of the alginate beads by the end of the reaction [100]. The optimization of immobilization parameters can reduce the leaching of small proteins, for example, by controlling the amount of alginate and the concentration of Ca2+ to fine tune the gel properties [101]. AnotherFigureFigure 18.18. strategy Chemo-enzymaticChemo-enzymatic to avoid leaching epoxidation is to of αuse-pinene the alginate catalyzed in by combination the lipaselipase fromfrom with AspergillusAspergillus other polymers, nigerniger supportsimmobilizedimmobilized or nanoparticles. inin calciumcalcium alginatealginateFor example, beads.beads. the lipase from oleaginous seeds of Pachira aquatica was immobilized by entrapment using a mixture of alginate and polyvinyl alcohol (PVA). The thermal The pores of the reticulated alginate network are large enough that small molecular weight stabilityAlginate increased, has also with been the lipaseused tokeeping prepare 60% gels of coitsnjugated original withactivity carrageenan, after 4 h at a50 sulfated °C, whereas galactan no molecules (<20 kDa) can diffuse into and out of the alginate beads [99]. A disadvantage of this support activitythat occurs was in detected numerous after species incubation of red of mari the nefree algae enzyme [103]. under For example, these conditions. the lipase Nevertheless, from Burkholderia this is that small proteins may leach into the bulk medium. For example, a crude extract containing lipases procedurecepacia was did first not crosslinked prevent the with loss glutaraldehyde, of activity of th ande biocatalyst, then it was given entrapped that its in activity gel beads decreased containing by of Yarrowia lipolytica was immobilized by microencapsulation in alginate [100]. The immobilization 50%equal after amounts five successive of calcium cycles alginate of hydrolysis and κ-carrageenan. of p-nitrophenyl The immobilized palmitate lipase[102]. was used to hydrolyze itself was successful, with the immobilized biocatalyst initially having high hydrolytic activity against olive oil in n-hexane at 40 °C; after 10 cycles of reaction, the immobilized lipase had a residual activity p-nitrophenyl laurate; however, 50% of the activity had leached out of the alginate beads by the end of the reaction [100]. The optimization of immobilization parameters can reduce the leaching of small proteins, for example, by controlling the amount of alginate and the concentration of Ca2+ to fine tune the gel properties [101]. Another strategy to avoid leaching is to use the alginate in combination with other polymers, supports or nanoparticles. For example, the lipase from oleaginous seeds of Pachira aquatica was immobilized by entrapment using a mixture of alginate and polyvinyl alcohol (PVA). The thermal stability increased, with the lipase keeping 60% of its original activity after 4 h at 50 ◦C, whereas no activity was detected after incubation of the free enzyme under these conditions. Nevertheless, this procedure did not prevent the loss of activity of the biocatalyst, given that its activity decreased by 50% Figure 18. Chemo-enzymatic epoxidation of α-pinene catalyzed by the lipase from Aspergillus niger after five successive cycles of hydrolysis of p-nitrophenyl palmitate [102]. immobilized in calcium alginate beads.

Catalysts 2020, 10, 697 18 of 29

Alginate has also been used to prepare gels conjugated with carrageenan, a sulfated galactan that occurs in numerous species of red marine algae [103]. For example, the lipase from Burkholderia cepacia was first crosslinked with glutaraldehyde, and then it was entrapped in gel beads containing equal amounts of calcium alginate and κ-carrageenan. The immobilized lipase was used to hydrolyze olive Catalysts 2020, 10, x FOR PEER REVIEW 18 of 29 oil in n-hexane at 40 ◦C; after 10 cycles of reaction, the immobilized lipase had a residual activity of of75% 75% [103 [103].]. A A similar similar strategy strategy was was used used to to immobilizeimmobilize thethe lipaselipase from Tsukamurella tyrosinosolvents E105 [104]. [104]. The The lipase lipase was was encapsulated encapsulated within within a mixture a mixture of sodium of sodium alginate alginate and carrageenan, and carrageenan, and thenand thencrosslinked crosslinked with glutaraldehyde. with glutaraldehyde. The biocatalys The biocatalystt was used was to usedcatalyse tocatalyse the resolution the resolution of racemic of (racemicR,S)-ethyl (R ,2-(2-oxopyrrolidinS)-ethyl 2-(2-oxopyrrolidin-1-yl)-1-yl) butyrate butyrate to (S)-ethyl-2-(2-oxopyrrolidin-1-yl) to (S)-ethyl-2-(2-oxopyrrolidin-1-yl) butyric acid butyric (Figure acid 19),(Figure an important 19), an important precursor precursor of an antiepileptic of an antiepileptic drug. The drug. hydrolysis The hydrolysis was performed was performed in Tris-HCl in Tris-HCl buffer (pHbuff er7.5) (pH at 35 7.5) °C at for 35 72◦C h, for with 72 a h, conversion with a conversion of 46% and of 46%an enantiomeric and an enantiomeric excess of excessproduct of of product 98.5%. Theof 98.5%. biocatalyst The biocatalyst was used wasin seven used insuccessive seven successive cycles of cycles reaction of reactionand maintained and maintained 64% of 64%its initial of its activity.initial activity. Recently, aa systemsystem was was described described in in which which lipase lipase M fromM fromMucor Mucor javanicus javanicuswas was first first adsorbed adsorbed onto ontocolloidal colloidal cationic cationic lignin lignin nanoparticles nanoparticles and then and coated then withcoated calcium with alginate.calcium alginate. The biocatalyst The biocatalyst was used wasin the used synthesis in the ofsynthesis butyl butyrate of butyl in bu a biphasictyrate in systema biphasic (10% systemn-hexane (10%/90% n-hexane/90% water). Both water). 1-butanol Both and 1- butanolbutyric acidand arebutyric suitable acid substrates are suitable for evaluatingsubstrates esterificationfor evaluating in aqueousesterification media, in becauseaqueous the media, butyl becausebutyrate the produced butyl butyrate is insoluble produced in water, is insoluble and is extracted in water, into and the is organic extracted phase into containing the organicn-hexane. phase containingThe conversion n-hexane. to n-butyl The conversion butyrate was to 77%n-butyl in 96butyrate h at 40 was◦C[ 10577%]. in 96 h at 40 °C [105]. Chemical modificationmodification of of alginates alginates is usedis used to improve to improve their properties,their properties, for example, for example, the oxidative the oxidativecleavage ofcleavage the uronic of the acid uronic units acid with units sodium with sodium periodate periodate in an acidin an medium, acid medium, which which results results in a inpolyaldehyde a polyaldehyde chain chain [106 [106].]. Hou Hou et al.et al. [107 [107]] used used a carriera carrier based based on on Fe Fe3O3O44 magneticmagnetic nanoparticles modifiedmodified with polydopamine andand alginatealginate polyaldehyde polyaldehyde for for the the immobilization immobilization of of CRL. CRL. Compared Compared to tothe the free free lipase, lipase, the the immobilized immobilized derivative derivative had had improved improved thermal thermal stability, stability, maintaining maintaining 60% of 60% initial of initialactivity activity in the rangein the ofrange 20 to of 60 20◦C to after 60 °C 30 after min of30incubation. min of incubation. The biocatalyst The biocatalyst maintained maintained a residual a residualactivity ofactivity 77% after of 77% 12 consecutiveafter 12 consecutive reuses. However,reuses. However, the performance the performance of the biocatalyst of the biocatalyst in organic in organicmedia was media not was evaluated. not evaluated. Although the use of alginate alginate is is traditional traditional in in the the immobilization immobilization of lipases lipases by by entrapment, entrapment, some some drawbacks are are associated with with the the use use of of this biomat biomaterial:erial: (i) (i) high high viscosity viscosity of of the the alginate alginate solution, solution, which makesmakes itit didifficultfficult to to form form a uniforma uniform gel; gel; (ii) (ii) low low mechanical mechanical strength, strength, which which means means that the that beads the beadsare susceptible are susceptible to breakage to breakage when subjected when subjected to shear to forces, shear liberating forces, liberating the enzyme the from enzyme the interiorfrom the of interiorthe bead of into the the bead bulk into reaction the bulk medium reaction and medium (iii) restricted and (iii) diff usionrestricted of substrates diffusion and of productssubstrates in and the productsalginate matrix, in the alginate resulting matrix, in low resulting activities in [61 low]. activities [61].

Figure 19. Enantioselective hydrolysishydrolysis of of ( R(R,S)-ethyl,S)-ethyl 2-(2-oxopyrrolidin-1-yl) 2-(2-oxopyrrolidin-1-y butyratel) butyrate catalyzed catalyzed by theby thelipase lipase from fromTsukamurella Tsukamurella tyrosinosolvents tyrosinosolventsE105 E105 immobilized immobilized in calcium in calcium alginate alginate/carrageenan/carrageenan beads. beads.

8. Other Biomaterials 8. Other Biomaterials Other polysaccharides have also been explored as supports for enzyme immobilization, such as Other polysaccharides have also been explored as supports for enzyme immobilization, such as gelatin, dextran, xanthan and pectin, as was recently reviewed by Bilal et al. [61]. Poly-hydroxybutyrate gelatin, dextran, xanthan and pectin, as was recently reviewed by Bilal et al. [61]. Poly- (PHB), an energy reserve polymer produced by several microorganisms, stands out [108]. It is hydroxybutyrate (PHB), an energy reserve polymer produced by several microorganisms, stands out hydrophobic and insoluble in organic solvents frequently used in synthesis reactions. Diﬀerent lipases [108]. It is hydrophobic and insoluble in organic solvents frequently used in synthesis reactions. Different lipases were immobilized on PHB and the immobilized derivatives were successfully used to produce esters by esterification and transesterification [108–111]. Another very useful polysaccharide is dextran, a complex branched glucan made by polymerization of α-D-glucopyranosyl units (Figure 20A) [112]. It has a large number of hydroxyl groups that can be chemically modified, for example by etherification and oxidation. Functionalized dextrins have been applied successfully to enhance activity and stability of various enzymes [113,114]. For example, aldehyde dextran has been used to modify free or immobilized lipases by

Catalysts 2020, 10, x FOR PEER REVIEW 19 of 29 Catalysts 2020, 10, 697 19 of 29 intramolecular covalent crosslinking, and to derivatize supports for immobilization [115,116]. Polyaldehyde dextran was used to derivatize a macroporous resin containing amino groups for wereimmobilization immobilized of onCRL. PHB The and biocatalyst the immobilized was applied derivatives in the were esterification successfully of used oleic to acid produce withesters oleic byalcohol, esterification in a solvent-free and transesterification system. The best [108 conversion–111]. was 92% in 12 h, at 40 °C. The biocatalyst was usedAnother in eight verysuccessive useful reaction polysaccharide cycles, ismaintaining dextran, a complex over 86% branched conversion glucan in the made last by cycle polymerization [117]. Tahir ofet αal.-d [118]-glucopyranosyl functionalized units dextran (Figure using 20A) 5-chloro-1-pentyne [112]. It has a large to number obtain ofO-pentinyl hydroxyl groupsdextran that(Figure can be20B). chemically This biomaterial modified, was for used example as a by support etherification for the andimmobilization oxidation. Functionalized of the lipase from dextrins Rhizopus have beenarrhizus applied by hydrophobic successfully adsorption to enhance and activity the immobilized and stability enzyme of various was enzymes applied [ 113in the,114 esterification]. For example, of aldehydeoctanoic acid dextran with hasgeraniol been in used n-hexane. to modify The freemaximum or immobilized conversion lipases was 90% by intramolecularin 160 h at 21 °C covalent [118]. crosslinking,Recently, and the to derivatizeuse of derivatized supports for immobilizationpullulan has [increased115,116]. Polyaldehydein the pharmaceutical dextran was usedand tobiotechnological derivatize a macroporous fields [119]. resin Pullulan containing is a fungal amino polysaccharide groups for immobilization consisting of maltotriose of CRL. The units biocatalyst (three wasglucose applied units in linked the esterification by α-1,4 glycosidic of oleic bonds) acid with connected oleic alcohol, by α-(1,6) in aglycosidic solvent-free bonds. system. Xu et The al. [120] best conversionimmobilized was a lipase 92% from in 12 Burkholderia h, at 40 ◦C. cepacia The biocatalyst by ionic adsorption was used inonto eight spherical successive particles reaction of pullulan cycles, maintainingacetate modified over with 86% succinic conversion anhydride in the last (Figure cycle 21). [117 The]. Tahir immobilized et al. [118 lipase] functionalized was applied dextran in the usingresolution 5-chloro-1-pentyne of (R,S)-1-phenylethanol to obtain O in-pentinyl n-heptane, dextran with (Figurea conversion 20B). Thisto (R biomaterial)-1-phenylethyl was acetate used as of a support50%, in 2 for h at the 45 immobilization°C, with an enantiomeric of the lipase excess from ofRhizopus the substrate arrhizus of 99%.by hydrophobic The immobilized adsorption lipase andalso thehad immobilized good operational enzyme stability; was applied after being in the used esterification for 10 cycles, of octanoic it still retained acid with over geraniol 80% of in itsn -hexane.original Theactivity maximum [120]. conversion was 90% in 160 h at 21 ◦C[118].

Figure 20. (A) Schematic representation of the chemical structure of the dextran (α-dD-glucopyranosyl units). ((BB)) SchematicSchematic representation representation of of the the chemical chemical structure structure of theof theO-pentynyl O-pentynyl dextran. dextran. The lightThe light pink circlespink circles randomly randomly highlight highlight the hydroxyl the hydroxyl groups groups that can that be can chemically be chemically modiﬁed. modified.

Recently,Natural materials the use of that derivatized are generally pullulan classified has increased as agro-industrial in the pharmaceutical wastes have and also biotechnological been used to ﬁeldsimmobilize [119]. Pullulanlipases. For is a fungalexample, polysaccharide sugarcane bagasse consisting has of been maltotriose used as units a support (three glucose for in unitssitu linkedimmobilization by α-1,4 glycosidicof lipases produced bonds) connected in solid-state by α -(1,6)fermentation, glycosidic as bonds.recently Xu reviewed et al. [120 by] immobilizedKrieger et al. a[121]. lipase Corn from cobs,Burkholderia rice husks, cepacia bananaby stalks, ionic coconu adsorptiont husks onto [122], spherical and loofah particles sponges of pullulan have also acetate been

Catalysts 2020, 10, x FOR PEER REVIEW 20 of 29 used. For example, rice husk (RH) was oxidized with NaIO4, functionalized with the spacer hexamethylenediamine (HMDA), and activated with glutaraldehyde, then CALB was immobilized covalently [123]. The resulting CALB-RH was used to catalyze the polycondensation of dimethylitaconate and 1,4-butanediol in solvent-free medium. The best conversion of Catalystsdimethylitaconate2020, 10, 697 to polyester was 92% in 72 h at 50 °C and 70 mbar [123]. This enzyme-based20 of 29 polymerization is undertaken at low temperatures and, therefore, maintains the vinyl groups intact, making them available for subsequent modifications; chemical polymerization is undertaken at modiﬁed with succinic anhydride (Figure 21). The immobilized lipase was applied in the resolution of temperatures above 150 °C, in which the vinyl group of the itaconic acid reacts, leading to undesirable (R,S)-1-phenylethanol in n-heptane, with a conversion to (R)-1-phenylethyl acetate of 50%, in 2 h at radical isomerization and crosslinking [123]. As another example, loofah sponge was used to 45 ◦C, with an enantiomeric excess of the substrate of 99%. The immobilized lipase also had good immobilize BCL by adsorption and used in the synthesis of 1-octyl acetate, with acetonitrile as the operational stability; after being used for 10 cycles, it still retained over 80% of its original activity [120]. solvent; the conversion was 94% in 24 h at room temperature [91].

Figure 21. Schematic representation of the chemical structurestructure ofof succinylatedsuccinylated pullulanpullulan acetate.acetate. The backbone of pullulan is a linear polymer of α-(1,6)-maltotriose.

9. ProsNatural and Cons materials of Using that Biopolymers are generally to Immobilize classiﬁed asLipases agro-industrial wastes have also been used to immobilize lipases. For example, sugarcane bagasse has been used as a support for The main advantages of using biopolymers like agarose, chitin, chitosan, cellulose and starch to in situ immobilization of lipases produced in solid-state fermentation, as recently reviewed by immobilize lipases are that they are sustainable and abundant resources, non-toxic and Krieger et al. [121]. Corn cobs, rice husks, banana stalks, coconut husks [122], and loofah sponges have also been used. For example, rice husk (RH) was oxidized with NaIO4, functionalized with the spacer hexamethylenediamine (HMDA), and activated with glutaraldehyde, then CALB was immobilized covalently [123]. The resulting CALB-RH was used to catalyze the polycondensation of dimethylitaconate and 1,4-butanediol in solvent-free medium. The best conversion of dimethylitaconate Catalysts 2020, 10, 697 21 of 29

to polyester was 92% in 72 h at 50 ◦C and 70 mbar [123]. This enzyme-based polymerization is undertaken at low temperatures and, therefore, maintains the vinyl groups intact, making them available for subsequent modiﬁcations; chemical polymerization is undertaken at temperatures above 150 ◦C, in which the vinyl group of the itaconic acid reacts, leading to undesirable radical isomerization and crosslinking [123]. As another example, loofah sponge was used to immobilize BCL by adsorption and used in the synthesis of 1-octyl acetate, with acetonitrile as the solvent; the conversion was 94% in 24 h at room temperature [91].

9. Pros and Cons of Using Biopolymers to Immobilize Lipases The main advantages of using biopolymers like agarose, chitin, chitosan, cellulose and starch to immobilize lipases are that they are sustainable and abundant resources, non-toxic and biocompatible. They are also easy to modify, due to the abundance of reactive groups on their surfaces. They can be used in a variety of forms, including gels, beads, and films, and can be used with a range of immobilization methods, such as entrapment, adsorption, and covalent bonding. Consequently, it is possible to produce biocatalysts with a range of different shapes and properties. Biopolymers also have some drawbacks as supports for the immobilization of lipases, although many of these drawbacks are common to other types of support. The gels, beads and films that are produced with these biopolymers often have poor mechanical stabilities that make them unsuitable for industrial application. For instance, the shear stress caused by agitation in batch bioreactors used for biodiesel synthesis may disrupt the support [124]. Additionally, in the case of immobilization by entrapment or by simple adsorption, the lipase may leach from the matrix, leading to a loss of activity and to poor reusability of the immobilized preparation. Some biopolymers, such as chitosan, have low chemical stability. In fact, the poor long-term stability of chitosan is still a major impediment to its use in large-scale applications. Since lipases are typically immobilized to allow their reuse, these drawbacks limit the industrial application of lipases immobilized on biopolymers. The reusability of immobilized lipases is normally determined in terms of residual activity, but the reasons for the loss of activity strongly depends on the support activation, immobilization mechanism and reaction media. In aqueous media, activity will typically be lost by the lipase leaching from the support if it is immobilized by ionic adsorption but in case of hydrophobic adsorption the trend of the lipase is stilling adsorbed on activated hydrophobic supports. In water-restricted media, such as organic solvents, lipases adsorbed on activated hydrophobic supports can be desorbed but not in case of ionic adsorption. The leakage of the lipases from the support has to be added to a distortion mechanism capable to inactivate the immobilized lipase. In fact, in case of supports capable to immobilize through covalent linkages, this is the only mechanism for the deactivation of the catalyst. Additionally, part of the immobilized preparation can be lost during the recovery step between reaction cycles. The problems of enzyme leaching and poor mechanical stabilities of biomaterials have been addressed through the use of cross-linking agents such as glutaraldehyde [41,58] and epichlorohydrin [38]. Another strategy to increase the mechanical resistance is to use composite materials, which may contain different biopolymers [125–127], inorganic materials or magnetic nanoparticles [14]. The use of biopolymers to immobilize lipases needs to be carefully thought out, since many biopolymers, in their original form, are highly hydrophilic, while lipases tend to perform better when immobilized on hydrophobic surfaces. For this reason, biomaterials used for the immobilization of lipases are often modified by the addition of groups that lead to more hydrophobic preparations. This is the case of cellulose paper functionalized with methacryloxy groups [82], and hydrophobic chitosan microspheres prepared by reductive amination using dodecyl aldehyde [76]. An additional consideration is that hydrophilic biomaterials can favor partitioning, leading to a microenvironment that can negatively influence the success of the immobilization and the performance of the lipase [128,129]. For example, in the production of biodiesel by esterification or transesterification, the alcohol substrate, Catalysts 2020, 10, 697 22 of 29 and the water that is generated in the reaction are highly hydrophilic as is the product glycerol, in the case of transesterification. The alcohol can partition onto hydrophilic supports [128,129], leading to a high concentration in the microenvironment of the lipase causing competitive inhibition or even denaturation [124,130] Glycerol can also partition onto hydrophilic supports, where it forms a layer that hinders mass transfer. Finally, interactions between hydrophilic supports and the medium may lead to different substrate concentrations in the bulk solution and in the microenvironment of the enzyme, possibly promoting phase separation and making it difficult to describe the reaction kinetics [131,132]. Relatively little attention has been given to the microenvironment of lipases immobilized on hydrophilic supports. The use of advanced analytic strategies, such as opto-chemical sensing [133], could allow better characterization of the behavior of lipases in these microenvironments.

10. Conclusions In this review, we have summarized and discussed the latest advances in the methods and strategies involved in the immobilization of lipases on biomaterials. There are many possibilities in the use of this type of material to immobilize and stabilize lipases of industrial interest, following simple protocols. Furthermore, the use of renewable materials provides an opportunity to develop processes that are more sustainable and economical, making the biocatalysts more competitive for use in industry. However, there are still some issues to be addressed before biocatalysts based on these biomaterials can be applied at large scale, especially in non-conventional media. For example, although many papers have reported lipase immobilization in biomaterials, in most cases the biocatalysts have only been applied in synthesis reactions at laboratory scale. Thus, there is a need for studies to evaluate the behavior of the processes at large scale. Issues such as the effect of shear stress on the integrity of the immobilized preparation and the regeneration of the support matrix after use need to be addressed, so that the support can be reused in many cycles. It is important to realize that, for the biocatalyst to be competitive in industry, it is not sufficient for it to last for 5 or 10 cycles (the typical duration of laboratory studies into reuse), it must last much, much longer. Additionally, it would be desirable to have a better understanding of the phenomena occurring during reactions with immobilized lipases in complex non-conventional media, including the sorption of solvents, reagents, products, and enzymes on the support matrix. Finally, a continuing challenge with respect to the use of new biopolymeric matrices lies in the identification of new biopolymers that have properties superior to those of existing biomaterials. For example, the number of polysaccharides available on the market is limited when compared to the large number of polysaccharides whose structures have been published or patented.

Author Contributions: R.C.A. and L.A.d.S. analyzed the data, wrote and revised the manuscript. J.M.d.A. contributed in the analysis of data and partially wrote the paper. N.K. wrote and revised the manuscript. C.M. designed, analyzed the data, wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Spanish Government projects (AGL2017-84614-C2-1-R), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico-CBAB/CABBIO-Brazilian-Argentine Center for Biotechnology, grant No:441015/2016-6) and FAPESP (São Paulo Research Foundation, grant No: 2018/07522-6) for financial support. Research scholarships were granted to R.C.A. by FAPESP (Grant No: 2020/00081-4), to J.M.A. and N.K. (grant No 303287/2019-5 by CNPq, and to L.A.S. and R.C.A. by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), a Brazilian government agency for the development of personnel in higher education. This study was financed in part by CAPES-Finance Code 001. Acknowledgments: The authors thank David A. Mitchell for help with the English expression. Conflicts of Interest: The authors declare that they have no conflict of interest.

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