inclusion and release of proteins from polysaccharide-based polyion complexes

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L Advanced Drug Delivery Reviews 31 (1998) 223–246 Inclusion and release of proteins from polysaccharide-based polyion complexes * Severian Dumitriu , Esteban Chornet University of Sherbrooke, Departement of Chemical Engineering, Sherbrooke, PQ, J1K 2R1, Canada Abstract The notion of a polyelectrolyte complex is well established for the complexation of two polymers one anionic, the other cationic. Electronic microscopy studies have shown the formation of a fibrilar structure. A method for the preparation of polyionic hydrogels from the complexation of chitosan and xanthan is reported. Electronic microscopy studies have shown the formation of a fibrilar structure. Stable hydrogels have been used to immobilize xylanase, lipase and protease. The immobilized xylanase and lipase activity was significantly higher than that of the free enzyme. 1998 Elsevier Science B.V. Keywords: Polyelectrolyte complex; Complexation; Chitosan; Xanthan Contents 1. Introduction ............................................................................................................................................................................ 223 2. Polyelectrolyte complex—recent development ........................................................................................................................... 224 2.1. Polyelectrolyte complex between natural polymers ............................................................................................................. 224 2.2. Polyelectrolyte complex between a natural and a synthetic polymer ..................................................................................... 226 2.3. Polyelectrolyte complex between synthetic polymers .......................................................................................................... 227 2.4. Complex formation between polyions and surfactants ......................................................................................................... 228 2.5. Potential uses of polyelectrolyte complex........................................................................................................................... 228 2.5.1. Polyelectrolyte-complex membrane ......................................................................................................................... 228 3. Protein–polyelectrolyte complexation ....................................................................................................................................... 229 3.1. Protein separation by precipitation..................................................................................................................................... 229 3.2. Protein separation by coacervation .................................................................................................................................... 230 3.3. Immobilization of enzymes ............................................................................................................................................... 230 4. Electrostatic interaction between polyions and nucleic acids ....................................................................................................... 240 References .................................................................................................................................................................................. 241 1. Introduction as hydrogen bonding, Coulomb forces, van der Waals forces, and transfer forces. Biofunctions such as gene Macromolecular complexes of different polymers information, and antigen–antibody reactions are are bound through intermolecular interactions, such based principally on the complexation of biopoly- mers such as proteins, polysaccharides and nucleic * acids. Corresponding author. Fax: (819) 821 7955; e-mail: [email protected] According to the nature of interactions the com- 0169-409X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0169-409X(97)00120-8

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Page 1: Inclusion and release of proteins from polysaccharide-based polyion complexes

LAdvanced Drug Delivery Reviews 31 (1998) 223–246

Inclusion and release of proteins from polysaccharide-based polyioncomplexes

*Severian Dumitriu , Esteban ChornetUniversity of Sherbrooke, Departement of Chemical Engineering, Sherbrooke, PQ, J1K 2R1, Canada

Abstract

The notion of a polyelectrolyte complex is well established for the complexation of two polymers one anionic, the othercationic. Electronic microscopy studies have shown the formation of a fibrilar structure. A method for the preparation ofpolyionic hydrogels from the complexation of chitosan and xanthan is reported. Electronic microscopy studies have shownthe formation of a fibrilar structure. Stable hydrogels have been used to immobilize xylanase, lipase and protease. Theimmobilized xylanase and lipase activity was significantly higher than that of the free enzyme. 1998 Elsevier ScienceB.V.

Keywords: Polyelectrolyte complex; Complexation; Chitosan; Xanthan

Contents

1. Introduction ............................................................................................................................................................................ 2232. Polyelectrolyte complex—recent development........................................................................................................................... 224

2.1. Polyelectrolyte complex between natural polymers ............................................................................................................. 2242.2. Polyelectrolyte complex between a natural and a synthetic polymer ..................................................................................... 2262.3. Polyelectrolyte complex between synthetic polymers .......................................................................................................... 2272.4. Complex formation between polyions and surfactants ......................................................................................................... 2282.5. Potential uses of polyelectrolyte complex........................................................................................................................... 228

2.5.1. Polyelectrolyte-complex membrane ......................................................................................................................... 2283. Protein–polyelectrolyte complexation ....................................................................................................................................... 229

3.1. Protein separation by precipitation..................................................................................................................................... 2293.2. Protein separation by coacervation .................................................................................................................................... 2303.3. Immobilization of enzymes ............................................................................................................................................... 230

4. Electrostatic interaction between polyions and nucleic acids ....................................................................................................... 240References .................................................................................................................................................................................. 241

1. Introduction as hydrogen bonding, Coulomb forces, van der Waalsforces, and transfer forces. Biofunctions such as gene

Macromolecular complexes of different polymers information, and antigen–antibody reactions areare bound through intermolecular interactions, such based principally on the complexation of biopoly-

mers such as proteins, polysaccharides and nucleic* acids.Corresponding author. Fax: (819) 821 7955; e-mail:

[email protected] According to the nature of interactions the com-

0169-409X/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PII S0169-409X( 97 )00120-8

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plexes can be divided into: (a) Polyelectrolyte com- lysozyme is required for catalysis of partially N-plexes; (b) charge-transfer complexes; (c) hydrogen- acetylated oligomers [27].bonding complexes; (d) stereocomplexes. Hyaluronan is involved in the development of

repair and disease processes by interacting withspecific binding proteins [28]. These proteins can beconveniently divided into those that are primarily

2. Polyelectrolyte complex—recent developmentextracellular, including aggrecan, link proteins, ver-sican, hyaluronectin and ‘receptors’ that are closely

The formation of complexes by the interaction ofassociated with the cell surface. The later category

oppositely-charged polyelectrolytes is well known. Aincludes a lymphocyte homing receptor and a re-

variety of polyelectrolyte complexes can be obtainedceptor that mediates cell locomotion.

by changing the chemical structure of componentMacromolecular interactions between negatively-

polymers, such as molecular weight, flexibility,and positively-charged proteins have been reported

functional group structure, charge density, hydro-to enhance functional properties including foaming

philicity and hydrophobicity balance, stereoregularity[29] and aggregation phenomena or gelation [30–

and compatibility, as well as reaction conditions: pH,33]. The interactions and amount of precipitation

ionic strength, concentration, mixing ratio, and tem-varied depending on the concentration of each

perature.protein in the mixture, the ionic strength and pH of

Potential fields of application of polyelectrolytethe solution [34].

complexes are: as membranes for different end usesInsoluble precipitates were formed in mixtures of

[1–3]; coatings on films and fibres [4]; implants forlysozyme and either a-lactalbumin or b-lactoglobu-

medical use [5–7]; microcapsules [8,9]; supports forlin [35]. Molecular modelling studies suggested

catalysts [10]; binding of pharmaceutical productsspecific electrostatic interactions between Glu-35 and

[11]; isolation and fractionation of proteins [12,13];Asp-53 in the catalytic sites of lysozyme, with Lys-

isolation of nucleic acid [14–16]. As well, the138 and Lys-141 at the dimerization site of b-

precipitation of nucleic acids with polyelectrolyteslactoglobulin [35].

has been investigated for Escherichia coli [15] andWhen soy protein was mixed with sodium algi-

wheatgerm [16].nate, the two polymers interacted to form electro-static complexes [36]. These interactions improved

2.1. Polyelectrolyte complex between natural the solubility and emulsifying activity.polymers We studied the complexation reaction between

xanthan and chitosan by determining the yields andChitosan has been used for the preparation of structure of the complexes formed as well as the

various polyelectrolyte-complex products with natu- swelling capacities. Three chitosan samples withral polyanions as carboxymethylcellulose [17], al- degrees of acetylation (DA) of 12, 16, and 19% wereginic acid [18], dextran sulfate [19,20], carbox- used. The complexation efficiency was as high asymethyldextran [21], heparin [22], carrageenan, pec- 90% of the available chitosan. The chitosan contenttin [23] and xanthan [24,25]. of the hydrogel depended upon the DA of chitosan

Colfen et al. [26] used, for the first time, analytical and, to a lesser extent, on the chitosan /xanthan ratioultracentrifugation to study the extent of complex used in the complexation reaction.formation between lysozyme and a deacetylated Scanning electron microscope (SEM) images ofchitosan (DA 5 1 and 0.1%). Sedimentation velocity the hydrogels are shown in Fig. 1 for typical gelexperiments gave clear evidence of interaction be- samples (32% chitosan, 68% xanthan). The imagestween chitosan (DA 5 1%) and lysozyme and an convey the message that the gels are porous and thatindication of interaction between lysozyme and the formation of fibrillar structures has occurred. Thechitosan (DA 5 0.1%). Lysozyme has six subsites, in channels present in the fibrillar gels have a pore size

27 26the active cleft each recognizing a sugar unit in a between 10 and 10 m (0.1–1 mm), whereas the27hexasaccharide sequence. Interaction of glucose-N- fibrils have a typical dimension of 10 m (100 nm).

acetyl amide residues with subsites C an E of The surface of the sphere has a homogenous porous

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Fig. 1. Scanning electron microscope images of typical gels (32% chitosan, 68% xanthan). (a) Image of cross section, 3 270; (b) image ofcross section, 3 8000; (c) image of external surface, 3 40 000; (d) image of external surface, 3 75 000; (e) image of internal section,3 40 000; (f) image of internal section, 3 60 000.

structure (Fig. 1d) which allows for the passage of Transmission electron microscope (TEM) imagespolymeric substrates to the regions where the im- of the hydrogels are shown in Fig. 2. TEM of themobilized enzymes are lodged. hydrogels shows: (a) a well-developed fibrillar struc-

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which leads to increased selectivity in the coacerva-tion step.

The complexation of papains with potassiumpoly(vinyl alcohol sulfate) (KPVS) as a function ofpH was studied using fluorescence spectroscopy[44,45]. The number of papain molecules bound toone molecule of KPVS should increase with pH (Fig.3). This is because: (a) the number of papain-at-tached, protonated basic groups increases with adecrease in pH; and (b) the state of dissociation of0503-groups in KPVS as the strong polyanion isunchanged over a wide range of pH.

Polyelectrolyte-complex formation betweenchitosan and polyacrilic acid has been previously

Fig. 2. Transmission electron microscope images of typical gels reported [46]. The composition of the complexes is a(32% chitosan, 68% xanthan). Image of cross section 3 60 000. function of the initial pH of the reaction mixture.

Skorikova et al., [47] used the potentiometricture with cavities in the 20 nm range and (b) a very method to study the reaction of formation of poly-uniform distribution of the hydrated swollen hydro- electrolyte complexes between chitosan and poly-gel following freezing, slicing and microscopic ex- acrylic acid, chitosan sulphate with poly-N,N-di-amination. methylaminoethylmethacrylate and 2,5-ionene bro-

mide. It was established that the composition of the2.2. Polyelectrolyte complex between a natural and polyelectrolyte complex depends on the pH of thea synthetic polymer medium.

The formation of a polyelectrolyte complex be-Formation of polymeric complexes of proteins tween heparin and aminoacetalized poly(vinyl al-

with synthetic polyelectrolytes are of interest to cohol) in aqueous media has been studied [48].simulate the intermolecular interactions during the Three conclusions were noted: (a) the heparin-amino-formation of biological systems.

The formation of complexes between proteins andsynthetic polyelectrolytes is evidenced by phaseseparation as a complex coacervate or a solidprecipitate. This is observed for potassium poly(vinylalcohol sulfate) and carboxyhemoglobin in the pres-ence of poly(dimethyldiallylammonium chloride)[37,38], lysozyme and poly(acrylic acid) [39], lyso-zyme and poly(methacrylic acid) [40], RNA poly-merase and poly(ethylene imine) [41], poly(di-methyldiallylammonium chloride) and bovine serumalbumin [42].

The interactions between proteins and syntheticpolyelectrolytes were investigated by turbidity[38,43], and quasielastic light-scattering techniques[43]. With the latter method, Park et al. [43] havebeen studying the interaction between strong polyca-tion poly(dimethyldiallylammonium chloride) andribonuclease, bovine serum albumin, and lysozyme. Fig. 3. Schematic illustration of the complexation between papainOf particular interest is the preferential binding to the and KPVS as a function of pH. s Papain molecule; ≠y KPVSpolycation of the lower-isoelectric-point proteins, molecule. From Ref. [44].

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acetalized poly(vinyl alcohol) complex is either in astate of liquid–solid equilibrium; (b) the reactionbetween heparin and aminoacetalized poly(vinylalcohol) is not perfectly stoichiometric; (c) theordered structure of heparin was disordered byformation of the complex at low pH.

Formation of a polyelectrolyte complex was in-vestigated as a function of pH using carboxymethylcellulose and poly(ethyleneimine). The formation ofpolyelectrolyte complex did not follow the stoichi-ometry [49].

Kikuchi and Kubota [50] have reported the struc-ture and properties of polyelectrolyte complexesconsisting of three materials: Polysaccharide (methylglycol chitosan), polypeptide (poly(sodium-L-gluta-mate)), and synthetic macromolecule [sulfate ofpoly(vinyl alcohol)]. The polyelectrolyte complexesprepared at pH 13.0 consist of two polymers (methylglycol chitosan and sulfate of poly(vinyl alcohol), atpH 2.0, 4.0 and 5.0 consist of the three materials,showing the helical structure of poly(sodium-L-gluta-

Fig. 4. Effect of the structure of polycations on the yield ofmate). At pH 6.0, 8.0 and 11.0, consist of the threepolyelectrolyte complexes. (a) QPVP–NaSS system, (b) 3X–materials with a random structure of poly(sosium-L-NaSS, (0) the NaSS solution was added to the polycation solution,

glutamate) [50]. (d) the polycation solution was added to the NaSS solution. 3Xintegral type polycation. From Ref. [56].

2.3. Polyelectrolyte complex between syntheticpolymers

The formation process of polyelectrolyte complex-Most of the work published in this area with es may be divided into three main classes (Fig. 5):

synthetic polyelectrolyte components was performed (1) primary complex formation; (2) formation pro-using conductometric, potentiometric or tubidimetric cess within intracomplexes; (3) intercomplex aggre-titration. Some synthetic polycations and polyanions gation process [57].used are presented in Table 1. The characteristics of The first step is realized through secondary bind-polyelectrolyte complexes between poly(sodium ing sources such as Coulomb forces (very rapid).styrene sulfonate) (NaSS) and a series of synthetic The second step involves the formation of new bondspolycations such as quaternized poly(4-vinyl and/or the correction of the distortions of thepyridine) (QPVP) (Fig. 4) have been described [57]. polymer chains. The third step involves the aggrega-

Table 1Synthetic polyanion and polycation used in polyelectrolyte complexation

Polyanion Polycation Refs.

Na-polyacrylate Poly(4-vinyl-N-butylpyridinium) bromide [50]Na-polystyrene sulfonate Poly(4-vinyl-N-butylpyridinium) bromide [50]K-poly(vinylalcohol) Poly(vinylamine)hydrochloride sulphate [51]Na-polystyrene sulfonate Poly(vinylbenzyltrimethyl)ammonium bromide [52]Polystyrene sulfonic acid Poly(vinylbenzyltrimethyl)ammonium hydroxide [52]Polyacrylic acid Various quaternized polycations [53]K-Polyvinyl sulphate Poly(dimethyldialkyl)ammmonium chloride [54]K-Poly(vinyl alcohol) Branched poly(ethylene imine) sulphate [55]

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Fig. 5. Schematic representation of the aggregation of polyelectrolyte complexes. From Ref. [56].

tion of secondary complexes, mainly through hydro- [68], dynamic light scattering [60,66,68,69], andphobic interactions [57]. ultrafiltration [66].

The preparation of three types of polyelectrolyte Polyelectrolyte–surfactant complexes made ofcomplexes formed between poly(vinylbenzyl- poly(styrenesulfonate) and different alkyltrimethyl-trimethylammonium chloride) and poly(methacrylic ammonium derivatives have been synthesized byacid) have been reported [58]. common precipitation in water [70]. Redissolved

The stoichiometry of the reactions between poly- in polar organic solvents, these complexes showcations (protonated polyethyleneimine, ionene, poly- polyelectrolyte behaviour. Cast films of these com-(vinylbenzyltrimethylammonium chloride) and poly- plexes exhibit highly ordered mesomorphous phase.anions (sodium polyacrylate, potassium poly- The variation of polyelectrolyte properties, as well asstyrenesulfonate) has been investigated [59]. It was that of surfactant properties, enables the fine-tuningfound that they reacted almost stoichiometrically to of phase morphologies, as well as the related me-give a polyelectrolyte complex. The complex showed chanical, electrical and optical properties [71].the sigmoid-type adsorption behaviour similar to theadsorption behaviour of a hydrophillic material. 2.5. Potential uses of polyelectrolyte complex

2.4. Complex formation between polyions and 2.5.1. Polyelectrolyte-complex membranesurfactants Membrane application of polyelectrolyte complex-

es has been widely developed, and there have beenPolymer–surfactant complexes have proved to be studies on the permeability of ions and low-molecu-

very interesting [60–64] because they offer intrigu- lar-weight solutes [72–75].ing similarities with biological assemblies. For ionic Chitosan in aqueous acid solution was surface-surfactants above the critical micelle concentration, reacted with polyanion aqueous solutions (heparin,the complexation is a consequence of the Coulombic sodium alginate, carboxymethylchitin, poly(acrylicinteraction of the polyion and the charged micelle acid) [76]. The chitosan–heparin complex film was[65–67]. stable in water and in saline solutions. It released

Dubin et al., have studied the soluble compl- heparin in blood plasma because of the competitionexes of sodium dodecyl sulfate (SDS) /Triton of fibrinogen and transferrin for heparin.X-100/poly(dimethyldiallylammonium chloride) Polyelectrolyte complex membranes were investi-(PDMAAC) by turbidimetry [68,69], viscosimetry gated in the systems glycol chitosan-poly(vinyl sul-

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fate) and methyl glycol chitosan-carboxymethyl)dex- peptide was promoted by the presence of poly-tran. The permeability of KCI, urea, and sucrose (ethyleneimine), while with the poly(L-lysine)-car-through the membrane was determined under various boxymethyl cellulose system, the pH-induced coil-pH [72]. The permeability in the neutral region was to-helix transition was scarcely affected by the2–10 times as high as that in acidic region. presence of carboxymethyl cellulose.

Affinity membrane based on chitosan were pre-pared by introducing group-specific ligands such as 3.1. Protein separation by precipitationreactive dyes, which mimic the conformation ofnicotinamide adenine dinucleotide and bind various Protein separation by polyelectrolytes is an attrac-kinases and dehydrogenases. tive process for protein purification and recovery. In

Chitosan microporous membranes were prepared this process the target proteins are obtained firstthrough the phase inversion method and subsequent- through selective protein–polyelectrolyte separation,ly coupled with Cibacron Blue F3GA to generate then recovered by adjusting pH or ionic strengthchitosan-dye affinity membrane [77]. This dye-cou- [96,126,127].pled membrane provides relatively large adsorption Protein precipitation by polyelectrolytes offerscapacity for human serum albumin. several advantages: high protein removal levels; the

feasibility of polyelectrolyte recycling; fractionalprecipitation of mixed proteins; more thermal stabili-ty of proteins complexing of ionic polysaccharides.

3. Protein–polyelectrolyte complexationThe success of fractionation is highly dependent onthe process pH.

Proteins interact strongly with both synthetic [78]The mechanism of protein–polyelectrolyte com-

and natural polyelectrolyte [79]. These interactionsplexes formation may be depicted as in Fig. 6

may result in amorphous precipitates [80–84], com-[44,45]. For the complexation of proteins (bovine

plex coacervate [85–88], gels [89], fibers [90] or theformation of soluble complexes [91–95].

The practical approach of the polyelectrolytecomplexation of proteins include:

1. Protein separation; protein recovery [96–101].2. Immobilization or stabilization of enzymes

[45,102–104].3. Modification of protein–substrate affinity [105–

107].4. Electrostatic interactions between proteins and

nucleic acids [108–113].

The efficiency of protein precipitation depends onseveral variables as: (a) the number and distributionof charged sites on the protein surface [43]; (b) thenature of the polyelectrolyte [114–116], (c) the pH ofsolution [117,118]; (d) ionic strength [119–121]; (e)polymer dosage [122–124].

The conformational changes of polypeptides asso-ciated with the formation of polyelectrolyte complex-es were studied for the systems of poly(L-glutamicacid), poly(ethyleneimine) and poly(L-lysine) [125].With the poly(L-glutamic acid)-poly(ethyleneimine) Fig. 6. The mechanisn of protein–polyelectrolyte complexessystem, the pH-induced helix formation of the poly- formation.

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serum albumin) and enzymes (ribonuclease, lyso- strength. Microcapsules composed of collagen andzyme) with both polycations an polyanions, Dubin et chondroitin sulfate were obtained by complexal., [43] proposed the formation of soluble complex coacervation and characterized by DSC, opticalprior to precipitation. microscopy, SEM, and UV–Vis spectroscopy [13].

Chen et al., [128] studied the mechanism of floc Collagen recovery, defined as the ratio of the amountformation in the system consisting of lysozyme of collagen in the microcapsules to the initial amountprecipitated with polyacrylic acid (PAA) of varying of collagen in the solution, was close to 100% withinmolecular weight. The mechanisms are summarized the range the concentration of chondroitin sulfateas below: 0.2–1.0% and with an optimal pH 5.

• Floc formation in lysozyme precipitation by high 3.3. Immobilization of enzymesmolecular weight (above roughly one million)PAA is governed by the polymer bridging mecha- Polyelectrolyte complexes can be successfullynism. used as a support for immobilization of enzymes.

• Floc formation in lysozyme precipitation by low It must be mentioned the large range of possiblemolecular weight (below several thousand) PAA utilizations of these systems in the treatment ofis governed by a charge neutralization mecha- residual waters (phenol degradation) and in thenism. production of ethanol. The polycation nature of

• With the decrease of polymer average molecular chitosan has led to its application in various fieldsweight, the mechanism of floc formation in [132], including encapsulation and immobilization oflysozyme precipitation gradually changes from microbial cells and mammalian cells [133,134].the polymer bridging in the charge neutralization Polyionic hydrogels, formed through interaction ofmechanism [128]. a polyanion (xanthan, carboxymetheylcellulose, al-

ginic acid) with a polycation (chitosan), have theClark and Glatz [122] have investigated the effect of advantage of creating an ionic microsystem whichpolyelectrolyte dosage and addition on the formation favours the stabilization of a protein polymer byof protein–polymer precipitates using egg white interacting with the free acid and base functions.protein and carboxymethylcellulose. Removal levels Moreover, this type of hydrogel has a good porousof 28% and 96% for total protein and lysozyme structure which facilitates diffusion of both therespectively, were obtained. No lysozyme activity substrate and the product of an enzymatic reactionwas lost as a result of the precipitation process [122]. [135–150]. Previous studies have shown that hydro-

gels obtained using xanthan and chitosan have a3.2. Protein separation by coacervation fibrous structure, good hydroscopic qualities and are

capable of immobilizing bioactive substances such asIn polyelectrolyte complex coacervation, anionic drugs or enzymes [24,151–153].

and cationic macromolecules form complexes and a We have immobilized protease (E.C. 3.4.2.1.19) inseparate dense liquid phase or coacervate [75,129]. a hydrogel obtained by complexation of xanthan with

Efficiency and selectivity are the factors that chitosan. The immobilization reaction yield variesinfluence the applicability of polyelectrolyte between 74 and 98.4%, this being a function of thecoacervation to protein separation [122,129,130]. concentration of protease dissolved in the xanthanProtein binding and subsequent coacervation depend solution (Fig. 7).on pH, ionic strength, protein /polyelectrolyte ratio, Proteases and xylanases have been coimmobilizedand polyelectrolyte molecular weight [129]. by inclusion in a xanthan–chitosan hydrogel. The

Selective phase separation with polyelectrolytes coimmobilization was carried out in order to obtaincan be used to separate a mixture of proteins [129]. an enzymatic system capable of hydrolyzing proteinThe selectivity of protein separation depends mainly and xylan remnants present in the wastewater of theon pH, increasing with pH, with molecular weight of food industry. Proteolytic activity is a function of thecationic polyelectrolyte [poly(diallyldimethylam- concentration of the amount of both protease andmonium chloride)] and with diminishing ionic xylanase that are coimmoblized. When the concen-

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Fig. 7. Variation of the immobilization yield as a function of enzyme concentration in the xanthan solution (0.65% w/w).

tration of xylanase is at 1% we have observed (Fig. decreases. The coimmobilization of protease and8) an increase of the protease activity as its con- xylanase thus shows a synergistic effect: thecentration increases to 1%, after which the activity proteolytic activity increases due to the presence of

Fig. 8. Variation of the protease activity coimmobilized with xylanase as a function of incubation time. [Xylanase] 5 1%; temperature 5

378C.

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xylanase (Fig. 9). The synergy observed on the matrix is saturated with xylanase. In this case anprotease activity by the xylanase causes an increase important quantity of xylanase remains in the hydro-of up to 85% for a ratio of protease /xylanase 5 1/1 gel which allows the protease, also free in the(g /g). This effect may be explained by the possi- system, to function. The proteolytic–xylanasic activi-bility that xanthan–chitosan–xylanase–protease in- ty is dependent on xylanase incubation time (Fig.teraction in the hydrogel stabilizes the protease 11). There is an important decrease in proteolyticstructure. A similar effect has been observed in the activity as incubation times for the xylanase reactioncoimmobilization of protease with peroxidase [154]. increase, especially over 20 min. During the sameAlso chitosan may activate immobilized enzymes as period, that is, over 20 min, the increase of xylanasehas been demonstrated for protease. The xylanase activity is only 8%. Under the conditions tested, theactivity in the protease–xylanase system is nega- optimal value is an incubation time of 10 min for thetively influenced by the presence of protease (Fig. xylanase reaction in order to obtain maximal10). For every tested xylanase concentration (in the proteolytic activity. During xylanasic incubation inpresence or absence of protease) there is an increase series the behaviour of the immobilized proteasein the xylanase activity as compared to the free does not change. Longer incubation times (Fig. 12)enzyme, but the protease present in the system do not cause inhibition by the substrate or by thereduces the xylanase activity without going below reaction products of the RBB–xylan that remain inthe activity of the free xylanase. This is observed the hydrogel.especially for the larger concentrations of xylanase The amount of activity of the immobilized xylan-used when the xanthan–chitosan–protease–xylanase ase is proportional to the enzyme concentration in

Fig. 9. Relative protease activity as a function of varying concentrations of coimmobilized xylanase. [Protease] 5 1%; incubation time 5 50min.

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Fig. 10. Relative xylanase activity as a function of varying concentrations of xylanase in the system. [Protease] 5 1%; substrate 5 RBB–xylan; incubation time 5 60 min; temperature 5 308C.

Fig. 11. Serial variation of protease and xylanase activities as a function of varying concentrations of xylanase in the hydrogel.[Protease] 5 1%; incubation time for xylanase reactions 5 60 min; temperature 5 308C; substrate 5 RBB–xylan; incubation time for proteasereactions 5 10 min; temperature 5 378C; substrate 5 hemoglobin.

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Fig. 12. Variations of relative protease and xylanase activities as a function of xylanase reaction incubation times. [Protease] 5 1%,[xylanase) 5 1%. Incubation times for protease reactions 5 50 min at 378C; substrate 5 hemoglobin.

Fig. 13. Variation of activity of immobilized or free xylanase as a function of temperature.

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the xanthan solution (Fig. 7). A maximal tempera- capable of hydrolyzing lipids and hemicellulosesture between 85–958C is found for immobilized present in food and industrial wastewaters.xylanase (Fig. 13) while for free xylanase this value The immobilized lipase is quite stable in this typeis between 40–508C. of hydrogel. The loss of activity is approximately

Images obtained by electronic microscopy of the 17% after 100 min of washing for samples con-xanthan–chitosan matrices, with or without xylanase, taining a high concentration of lipase (1.8%) andshow a fibrillar structure in which there are globular 1.8% for samples containing 0.35% lipase. Theformations (in the presence of xylanase) (Fig. 14). activity of the immobilized lipase in an emulsion ofThese globular formations are formed by the triple olive oil depends on reaction time temperature andcomplexation between xanthan–chitosan–xylanase. pH (Fig.s 15–17). According to Figs. 15–17 theIn a cross-section of the beads (Fig. 14c) we observe optimal conditions for lipase activity in aqueouslamellae of fibrils in a layer approximately 4 mm medium are: incubation time 5 10 min, incubationthick followed by an unorganized fibrous structure. temperature 5 378C, pH 5 7.5.

The immobilization of lipase and lipase–xylanase Lipase activity in isooctane has been studied as ain xanthan–chitosan hydrogels have been studied in function of the concentration of olive oil, incubationorder to determine the immobilization capacity of time and percentage of water in the system. Thethis type of gel and attempt to obtain a biocatalyst optimal time and temperature of incubation are

Fig. 14. Scanning electron microscopy of hydrogels with or without immobilized xylanase. (a) Exterior of the hydrogel xanthan–chitosanbeads, 3 40 000 (b) Exterior of the hydrogel xanthan–chitosan beads containing immobilized xylanase, [xylanase] 5 1.56%. (c) Interiorstructure of the hydrogel xanthan–chitosan beads, 3 40 000. (d) Interior structure of the hydrogel xanthan–chitosan beads containingimmobilized xylanase, (xylanase) 5 1.56%, 3 40 000.

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Fig. 15. Reaction rate for immobilized lipase in the polyionic hydrogel as a function of incubation time. Incubation temperature 5 378C,pH 5 7.5, substrate 5 olive oil emulsion.

Fig. 16. Reaction rate for immobilized lipase in the polyionic hydrogel as a function of incubation temperature. Incubation time 5 10 min,pH 5 7.5, substrate 5 olive oil emulsion.

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Fig. 17. Variation of the reaction rate for immobilized lipase in the polyionic hydrogel as a function of time. [Olive oil] 5 34.44%;temperature 5 348C; solvent 5 isooctan.

different then those derived for the aqueous emulsion methyl-N,N- diethylaminoethylacrylate and acrylam-of olive oil. Optimal time is 24 h and temperature is ide has been investigated by means of turbidimetry,348C. Optimal values for the reaction in organic light-scattering measurements, and determination ofmedium are: concentration of lipase in the the enzyme activity [104]. Only a fraction of thehydrogel 5 1.5%; [olive oil] 5 34.4%. The presence invertase molecules is engaged in Coulombic inter-of water in the isooctane favours the activity of the action and this fraction shows a rather small enzymeimmobilized lipase (Fig. 18). activity.

The coimmobilization of lipase with xylanase Heng and Glatz [154] have explored the use ofchanges the properties of both enzymes. For lipase charged fusions for selective recovery of b-galacto-we observe different kinetics with inhibition occur- sidase. The advantage of negatively-charged purifica-ring after 14 min of incubation (Fig. 19). Moreover, tion fusions is the change in the selectivity ofas the concentration of coimmobilized xylanase recovery via precipitation with the cationic polyelec-increases this causes a synergy in the lipase activity. trolyte [155]. The tails were a series of polyaspartateIn an organic medium the activity of the coimmobil- fusions adding 1, 5, 11 and 16 negative charges toized lipase decreases (Fig. 20). b-galactosidase [156]. Because active b-galactosi-

Xylanase activity, in a system coimmobilized with dase is a tetramer, each fusion contains four tails.lipase, is increased due to a synergistic effect from The b-galactosidase fusion tails have been used forthe lipase (Fig. 13). immobilization on ion-exchange membranes contain-

The interaction between invertase and poly(di- ing quaternary amine functionalities [157].methyldiallylammonium chloride), poly(N-methyl- The complexation of papain with potassium poly-N,N-diethylaminoethylacrylate) or copolymer of N- (vinyl alcohol) sulfate and sodium poly(styrene

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Fig. 18.Variation of the activity of the immobilized lipase in the presence of isooctane as a function of the volume of water introduced to thereaction. V 5 5000 ml; incubation time 5 24 h; [olive oil] 5 34.44%; temperature 5 348C.isooctane

Fig. 19. The relative activity of lipase as a function of the concentration of coimmobilized xylanase and incubation time. [Lipase] 5 1%;incubation temperature 5 378C; substrate 5 olive oil emulsion.

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Fig. 20. Lipase activity in the lipase–xylanase system in isooctane medium. [Xylanase] 5 1%; [olive oil] 5 20.27%.

sulfonate) was studied at different pH levels by plexes has been demonstrated for several enzymes inmeans of colloid titration [44,45]. The moles of the solution including alcohol oxidase, horseradish per-sulfate or sulfonate groups in the polyelectrolytes oxidase, lactate oxidase and glucose oxidase [160].that took part in the complexation with 1 g papain From the immobilized experiments there is evidencevaried depending on pH due to a pH-induced change that the protein–polyelectrolyte complexes also havein the protonation of the protein basic groups: 11amino (including one N-terminal), 2 imidazolyl, and12 guanidyl groups. A loss in the enzyme activitydue to complexation was observed. The structure ofthe protein /polyelectrolyte complexes is shownschematically in Fig. 21, where the complex consistsof a number of nonflexible and global proteinmolecules bridged or bundled by one loosely-extend-ed polyelectrolyte ion [158].

Complexation between a-chymotrypsin and theself-aggregate of a cholesterol-bearing pullulan wasstudied by size-exclusion column chromatography,fluorescence spectroscopy, circular dichroism, anddifferential scanning calorimetry [159]. Subsequentanalysis indicated that the thermal stability of a-chymotrypsin increases dramatically upon complex-ation with the cholesterol-bearing pullulan self-ag- Fig. 21. Structure of an intramolecular complex. The open andgregate. solid circles represent ionizable groups in the polyion and protein,

The formation of protein–polyelectrolyte com- respectively. From Ref. [157].

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the ability to improve the operational stability of theenzyme activity during catalysis.

The direct reaction between chitosan and alginatecreates the capsules with a bipolymer membranehaving certain porosity and mechanical strength[161,162].

Kim, and Kim [163] have demonstrated thebiocompatibility of the encapsulation process, thegrowth kinetics of encapsulated hybridoma cells(ATCC CRL-1606 and ATCC HB-8852) whichproduce the monoclonal antibodies against humanfibronectin and bovine lactoferrin, and monoclonal Fig. 22. A. Release of oxalate from cultured A. tricolor cells atantibody production kinetics. ATCC CRL-1606 and 1,3, and 5 days of treatment. A 5 freely suspended calls, B 5

HB-8852 grow well in the polyelectrolyte chitosan– calcium–alginate-gel entrapped cells, C 5 chitosan-gel entrappedcells, D 5 freely-suspended cells with 5 mg chitosan /100 mlalginate capsules, reaching maximum cell densitiesmedium; E 5 freely-suspended cells with 10 mg chitosan /100 mlof 8.2 and 2.8 3 107 cells per ml, respectively [163].medium. B. Protein concentration of medium filtrates of Asclepias

The concentration of the monoclonal antibodies syriaca L. cells entrapped in chitosan or calcium alginate gels 24,reached 507 and 106 mg/ml for ATCC CRL-1606 48, 72 and 96 h after immobilization. From Ref. [171].and HB-8852 respectively in the capsules. Theseconcentrations are about 20 times higher than theconcentrations of the monoclonal antibody produced [169]. The rate of the cell (chick embryo fibrobkasts,by free cell culture [163]. CEF) adhesion to the each collagen–chitosan coated

The eukaryotic microbial systems can be encapsu- dish was greater than to a collagen only coated dish.lated in polyelectrolyte complexes prepared from Only 10% addition of chitosan to the collagen matrixsodium cellulose sulfate and poly(dimethyldiallylam- improved the cell attachment to the matrix. Decreas-monium chloride) with maintenance of viability ing the ratio of chitosan in the collagen-chitosan[10,164]. matrix, the cell growth rate increased [169].

Forster et al. [165] have encapsulated Serratia Chitosan has been used as a concomitant im-marcescens B345 in these polyelectrolyte complex- mobilizing and permeabilizing agent for culturedes. This strain converts gluconic acid to 2-ketog- plant cells [170,171]. Chitosan was used as theluconic acid. Urease [166], invertase [9], cytochrome polyelectrolyte for gel formation and sodium tri-c, liver microsomes, and hemoglobin have also been phosphate as the multivalent counterion. In the casesuccessfully immobilized with this method [167]. of alginate gel, entrapped Amaranthus tricolor, and

Shioya et al. [168] have developed the encapsu- Asclepias syriaca L cells, sodium alginate andlated method based on the electrostatic interactions calcium chloride served as polyanion and counterionof chitosan as a polycation with sodium carboxy- respectively [172]. Release of oxalate, as well asmethylcellulose (CMC) as a polyanion. The ex- protein (Fig. 22a,b), from calcium-alginate-gel en-perimental results suggest that the salt concentration trapped cells was significantly lower than from(NaCl) in the chitosan solution and the molecular chitosan-gel entrapped cells [172].

5weight of chitosan (MW between 1.5 3 10 and62.8 3 10 ) may have a significant effect on the

permeability of the CMC-Chitosan capsule mem- 4. Electrostatic interaction between polyions andbrane [168]. Adding salt may cause a reduction in nucleic acidsthe effective charge by a shielding effect and thenpreventing the chitosan from reacting with CMC It is well known that chitosan inhibits the growth[168]. of a wide variety of bacteria. However, there are two

In order to improve the cell attachment to the proposed mechanisms for microbial growth inhibi-collagen matrix, chitosan which has a positive charge tion. One is that chitosan acts mainly on the surfacewas mixed with the collagen matrix in various ratios of the bacteria and interacts with the cell membrane

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