Enzyme and Microbial Technology 35 (2004) 126–139

Application of chitin- and chitosan-based materialsfor enzyme immobilizations: a review

Barbara Krajewska∗Jagiellonian University, Faculty of Chemistry, 30-060 Kraków, Ingardena 3, Poland

Received 11 September 2003; received in revised form 24 December 2003; accepted 24 December 2003

Abstract

As functional materials, chitin and chitosan offer a unique set of characteristics: biocompatibility, biodegradability to harmless products,nontoxicity, physiological inertness, antibacterial properties, heavy metal ions chelation, gel forming properties and hydrophilicity, andremarkable affinity to proteins. Owing to these characteristics, chitin- and chitosan-based materials, as yet underutilized, are predicted to bewidely exploited in the near future especially in environmentally benign applications in systems working in biological environments, amongothers as enzyme immobilization supports. This paper is a review of the literature on enzymes immobilized on chitin- and chitosan-basedmaterials, covering the last decade. One hundred fifty-eight papers on 63 immobilized enzymes for multiplicity of applications rangingfrom wine, sugar and fish industry, through organic compounds removal from wastewaters to sophisticated biosensors for both in situmeasurements of environmental pollutants and metabolite control in artificial organs, are reviewed.© 2004 Elsevier Inc. All rights reserved.

Keywords:Chitin; Chitosan; Enzyme immobilization; Applications; Review

1. Why enzymes?

While conventional methodologies of chemical processeshave been developed in the past decades to a level allow-ing production, separation and analytical determination ofan enormous range of sophisticated products, alternativemethodologies that are not only efficient and safe but alsoenvironmentally benign and resource- and energy-saving,are being increasingly sought. One of the most promisingstrategies to achieve these goals is the utilization of enzymes[1–5]. Enzymes exhibit a number of features that make theiruse advantageous as compared to conventional chemicalcatalysts. Foremost among them are a high level of catalyticefficiency, often far superior to chemical catalysts, and ahigh degree of specificity that allows them to discriminatenot only between reactions but also between substrates (sub-strate specificity), similar parts of molecules (regiospeci-ficity) and between optical isomers (stereospecificity).These specificities warrant that the catalyzed reaction is notperturbated by side-reactions, resulting in the productionof one wanted end-product, whereas production of undesir-able by-products is eliminated. This provides substantiallyhigher reaction yields reducing material costs. In addition,

∗ Tel.: +48 12 6336377; fax:+48 12 6340515.E-mail address:krajewsk@chemia.uj.edu.pl (B. Krajewska).

enzymes generally operate at mild conditions of temper-ature, pressure and pH with reaction rates of the order ofthose achieved by chemical catalysts at more extreme condi-tions. This makes for substantial process energy savings andreduced manufacturing costs. Also, enzymes practically donot present disposal problems since, being mostly proteinsand peptides, they are biodegradable and easily removedfrom contaminated streams. This unique set of advantageousfeatures of enzymes as catalysts has been exploited since the1960s and several enzyme-catalyzed processes have beensuccessfully introduced to industry, e.g. in the productionof certain foodstuffs, pharmaceuticals and agrochemicals,but now also increasingly to organic chemical synthesis.

2. Why immobilize enzymes?

In addition to the unquestionable advantages, there existsa number of practical problems in the use of enzymes. Tothese belong: the high cost of isolation and purification ofenzymes, the instability of their structures once they are iso-lated from their natural environments, and their sensitivityboth to process conditions other than the optimal ones, nor-mally narrow-ranged, and to trace levels of substances thatcan act as inhibitors. The latter two result in enzymes’ shortoperational lifetimes. Also, unlike conventional heteroge-

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2003.12.013

B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 127

neous chemical catalysts, most enzymes operate dissolvedin water in homogeneous catalysis systems, which is whythey contaminate the product and as a rule cannot be recov-ered in the active form from reaction mixtures for reuse.

Several methods have been proposed to overcome theselimitations, one of the most successful being enzyme im-mobilization [1–6]. Immobilization is achieved by fixingenzymes to or within solid supports, as a result of whichheterogeneous immobilized enzyme systems are obtained.By mimicking the natural mode of occurence in living cells,where enzymes for the most cases are attached to cellularmembranes, the systems stabilize the structure of enzymes,hence their activities. Thus, as compared to free enzymesin solution immobilized enzymes are more robust and moreresistant to environmental changes. More importantly, theheterogeneity of the immobilized enzyme systems allowseasy recovery of both enzyme and product, multiple reuseof enzymes, continuous operation of enzymatic processes,rapid termination of reactions and greater variety of biore-actor designs.

Enzymes may be immobilized by a variety of methods,which may be broadly classified as physical, where weak in-teractions between support and enzyme exist, and chemical,where covalent bonds are formed with the enzyme[1–4,6,7].To the physical methods belong: (i) containment of an en-zyme within a membrane reactor, (ii) adsorption (physi-cal, ionic) on a water-insoluble matrix, (iii) inclusion (orgel entrapment), (iv) microencapsulation with a solid mem-brane, (v) microencapsulation with a liquid membrane, and(vi) formation of enzymatic Langmuir-Blodgett films. Thechemical immobilization methods include: (i) covalent at-tachment to a water-insoluble matrix, (ii) crosslinking withuse of a multifunctional, low molecular weight reagent, and(iii) co-crosslinking with other neutral substances, e.g. pro-teins. Numerous other methods which are combinations ofthe ones listed or original and specific of a given supportor enzyme have been devised. However, no single methodand support is best for all enzymes and their applications.This is because of the widely different chemical characteris-tics and composition of enzymes, the different properties ofsubstrates and products, and the different uses to which theproduct can be applied. Besides, all of the methods presentadvantages and drawbacks. Adsorption is simple, cheap andeffective but frequently reversible, covalent attachment andcrosslinking are effective and durable, but expensive andeasily worsening the enzyme performance, and in mem-brane reactor-confinment, entrapment and microencapsula-tions diffusional problems are inherent. Consequently, as arule the optimal immobilization conditions for a chosen en-zyme and its application are found empirically by a processof trial and error in a way to ensure the highest possibleretention of activity of the enzyme, its operational stabilityand durability.

Advantageous though it is, the immobilization involves anumber of effects worsening the performance of enzymes[1–4,6,7]. Compared with the free enzyme, most commonly

the immobilized enzyme has its activity lowered and theMichaelis constant increased. These alterations result fromstructural changes introduced to the enzyme by the ap-plied immobilization procedure and from the creation ofa microenvironment in which the enzyme works, differentfrom the bulk solution. The latter is strongly dependent onthe reaction taking place, the nature of the support and onthe design of the reactor. Furthermore, being two phasesystems, the immobilized enzyme systems suffer from in-evitable mass transfer limitations, producing unfavourableeffects on their overall catalytic performances. These,however, may be reduced by applying appropriate reactordesigns.

For the implementation in a commercial process all bene-ficial and detrimental effects of whether a chemical catalystor an enzyme is chosen, and whether a free or immobilizedenzyme is used, have to be weighed taking into account allrelevant aspects, health and environmental included, in ad-dition to obvious economical viability. To date, several im-mobilized enzyme-based processes have proved economicand have been implemented on a larger scale, mainly inthe food industry, where they replace free enzyme-catalyzedprocesses, and in the manufacture of fine speciality chem-icals and pharmaceuticals, particularly where asymmetricsynthesis or resolution of enantiomers to produce opticallypure products are involved[1–5,8]. A selection of currentlyused immobilized-enzyme processes, in the approximate or-der of the decreasing scale of manufacture, is given inTable1. The scale of the processes ranges from about 106 t per yearfor high-fructose corn syrup, arguably one of the most com-mercially important immobilized enzyme-based process, toabout 102 t per year for enantiopurel-DOPA [5].

Areas of present and potential future applications of im-mobilized enzyme systems other than industrial (Table 1)include: laboratory scale organic synthesis, and analyticaland medical applications[1–5,7]. Having been shown to beable to catalyze reactions not only in aqueous solutions butalso in organic media, enzymes offer great potential for as-sisting organic synthesis[9]. They can simplify the chem-ical procedures by reducing the number of synthetic steps,they can enhance the purity of the products, and most impor-tantly, they can catalyze regio- and stereoselective synthesisgiving, otherwise unobtainable compounds with the desiredproperties.

In analytical applications immobilized enzymes are usedchiefly in biosensors[3,10–12]and to a lesser extent, in diag-nostic test strips. Biosensors are constructed by integratingbiological sensing systems, e.g. enzyme(s), with transduc-ers. These obtain a chemical signal produced by the interac-tion of the biological system with an analyte and transduceit into a measurable response. Different kinds of transduc-ers have been employed in biosensors, viz potentiometric,amperometric, conductometric, thermometric, optical andpiezo-electric, most of the current research being placed onthe first two. Enzymes for the most cases are immobilized ei-ther directly on a transducer’s working tip or in/on a polymer

128 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139

Table 1Some of the more important industrial applications of immobilized enzyme systems[1–3,5]

Enzyme (EC number) Substrate Product

Glucose isomerase (5.3.1.5) Glucose Fructose (high-fructose corn syrup)�-Galactosidase (3.2.1.23) Lactose Glucose and galactose (lactose-free milk and whey)Lipase (3.1.1.3) Triglycerides Cocoa butter substitutesNitrile hydratase (4.2.1.84) Acrylonitrile Acrylamide

3-Cyanopyridine NicotinamideAdiponitrile 5-Cyanovaleramide

Aminoacylase (3.5.1.14) d,l-Aminoacids l-Amino acids (methionine, alanine, phenylalanine, tryptophan, valine)Raffinase (3.2.1.22) Raffinose Galactose and sucrose (raffinose-free solutions)Invertase (3.2.1.26) Sucrose Glucose/fructose mixture (invert sugar)Aspartate ammonia-lyase (4.3.1.1) Ammonia+ fumaric acid l-Aspartic acid (used for production of synthetic sweetener aspartame)Thermolysin (3.4.24.27) Peptides AspartameGlucoamylase (3.2.1.3) Starch d-GlucosePapain (3.4.22.2) Proteins Removal of “chill haze” in beersHydantoinase (3.5.2.2) d,l-Amino acid hydantoins d,l-Amino acidsPenicillin amidase (3.5.1.11) Penicillins G and V 6-Aminopenicillanic acid (precursor of semi-synthetic penicillins,

e.g. ampicillin)�-Tyrosinase (4.1.99.2) Pyrocatechol l-DOPA

membrane tightly wrapping it up. In principle, due to en-zyme specificity and sensitivity biosensors can be tailoredfor nearly any target analyte, and these can be both enzymesubstrates and enzyme inhibitors. Advantageously, their de-termination is performed without special preparation of thesample. Meeting the demand for practical, cost-effective andportable analytical devices, enzyme-based biosensors haveenormous potential as useful tools in medicine, environmen-tal in situ and real time monitoring, bioprocess and food con-trol, and in biomedical and pharmaceutical analysis. Theiruse, impaired as yet by not quite satisfactory reliability, ispredicted to become widely accepted once their storage andoperational stabilities have been improved. The most exten-sively studied enzymes for the application in enzyme-basedbiosensors are presented inTable 2. Of these, glucose sen-sors are the most widely studied constituting ca. 1/3 of the

Table 2Some of the most frequently studied enzymes for enzyme-based biosensors[3,10–12]

Enzyme (EC number) Substrate Application

Glucose oxidase (1.1.3.4) Glucose Diagnosis and treatment of diabetes, food science, biotechnologyHorseradish peroxidase (1.11.1.7) H2O2 Biological and industrial applications, inhibition-based

determination of heavy metal ions and pesticidesLactate oxidase (1.13.12.4) Lactate Sports medicine, critical care, food science, biotechnologyTyrosinase (1.14.18.1) Phenols, polyphenols Determination of phenolic compounds in foods, inhibition-based

determination of carbamate pesticidesGlutamate oxidase (1.4.3.11) Glutamate Food science, biotechnologyUrease (3.5.1.5) Urea Medical diagnosis, artificial kidney, environmental monitoringAlcohol dehydrogenase (1.1.1.1) Ethanol Food science, biotechnologyAcetylcholinesterase (3.1.1.7) Acetylcholine, acetylthiocholine Inhibition-based determination of organophosphorus and carbamate

pesticidesCholine oxidase (1.1.3.17) Choline Enzyme used in conjunction with acetycholinesteraseLactate dehydrogenase (1.1.1.27) lactate Sports medicine, critical care, food science, biotechnologyCholesterol oxidase (1.1.3.6) Cholesterol Medical applicationsPenicillinase (3.5.2.6) Penicillins Pharmaceutical applicationsAlliinase (4.4.1.4) Cysteine sulfoxides Food industry (garlic-, onions- and leek-derived products)

enzyme-biosensors literature, the subsequent ten sensors oc-cupy another 1/3 of the literature and the other sensors theremaining 1/3[11]. From a practical and commercial pointof view, four of the sensors listed, namely glucose, lactate,urea and glutamate have been widely used[12].

Medical applications of immobilized enzymes include[1,4,13] diagnosis and treatment of diseases, among thoseenzyme replacement therapies, as well as artificial cells andorgans, and coating of artificial materials for better bio-compatibility. Offering a great potential in this area, realapplication of immobilized enzymes has as yet sufferedfrom serious problems from their toxicity to the human or-ganism, allergenic and immunological reactions as well asfrom their limited stability in vivo. Examples of potentialmedical uses of immobilized enzyme systems are listed inTable 3.

B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 129

Table 3Selected potential medical uses of immobilized enzymes[1,4,13]

Enzyme (EC number) Condition

Asparaginase (3.5.1.1) LeukemiaArginase (3.5.3.1) CancerUrease (3.5.1.5) Artificial kidney, uraemic disordersGlucose oxidase (1.1.3.4) Artificial pancreasCarbonate dehydratase (4.2.1.1)

+ catalase (1.11.1.6)Artificial lungs

Catalase (1.11.1.6) AcatalasemiaGlucoamylase (3.2.1.3) Glycogen storage diseaseGlucose-6-phosphate

dehydrogenase (1.1.1.49)Glucose-6-phosphate dehydrogenasedeficiency

Xanthine oxidase (1.1.3.22) Lesch–Nyhan diseasePhenylalanine ammonia lyase

(4.3.1.5)Phenylketonuria

Urate oxidase (1.7.3.3) HyperuricemiaHeparinase (4.2.2.7) Extracorporeal therapy procedures

3. Why immobilize enzymes on chitin- andchitosan-based materials?

The properties of immobilized enzymes are governed bythe properties of both the enzyme and the support material[4,6]. The interaction between the two lends an immobilizedenzyme specific physico-chemical and kinetic properties thatmay be decisive for its practical application, and thus, a sup-port judiciously chosen can significantly enhance the opera-tional performance of the immobilized system. Although it isrecognized that there is no universal support for all enzymesand their applications, a number of desirable characteristicsshould be common to any material considered for immo-bilizing enzymes. These include: high affinity to proteins,availability of reactive functional groups for direct reactionswith enzymes and for chemical modifications, hydrophilic-ity, mechanical stability and rigidity, regenerability, and easeof preparation in different geometrical configurations thatprovide the system with permeability and surface area suit-able for a chosen biotransformation. Understandably, forfood, pharmaceutical, medical and agricultural applications,nontoxicity and biocompatibility of the materials are alsorequired. Furthermore, to respond to the growing publichealth and environmental awareness, the materials should bebiodegradable, and to prove economical, inexpensive.

Of the many carriers that have been considered and stud-ied for immobilizing enzymes, organic or inorganic, naturalor synthetic, chitin and chitosan are of interest in that theyoffer most of the above characteristics.

Chitin and chitosan are natural polyaminosaccharides[14–28], chitin being one of the world’s most plenti-ful, renewable organic resources. A major constituentof the shells of crustaceans, the exoskeletons of insectsand the cell walls of fungi where it provides strengthand stability, chitin is estimated to be synthesized anddegraded in the biosphere in the vast amount of atleast 10 Gt each year. Chemically, chitin is composed of�(1 → 4) linked 2-acetamido-2-deoxy-�-d-glucose units

OOH

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HOO

OOH

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NHC=OCH3

NHC=OCH3

Chitin

NH2 NH2 NH2

Chitosan

OH OH OH

Cellulose

O

O

O

Fig. 1. Structure of chitin, chitosan and cellulose.

(or N-acetyl-d-glucosamine)[14], forming a long chainlinear polymer (Fig. 1). It is insoluble in most solvents.Chitosan, the principal derivative of chitin, is obtained byN-deacetylation to a varying extent that is characterized bythe degree of deacetylation, and is consequently a copoly-mer of N-acetyl-d-glucosamine andd-glucosamine. Chitinand chitosan can be chemically considered as analoguesof cellulose, in which the hydroxyl at carbon-2 has beenreplaced by acetamido and amino groups, respectively. Chi-tosan is insoluble in water, but the presence of amino groupsrenders it soluble in acidic solutions below pH about 6.5. Itis important to note that chitin and chitosan are not singlechemical entities, but vary in composition depending on theorigin and manufacture process. Chitosan can be defined aschitin sufficiently deacetylated to form soluble amine salts,the degree of deacetylation necessary to obtain a solubleproduct being 80–85% or higher.

Commercially, chitin and chitosan are obtained at a rel-atively low cost from shells of shellfish (mainly crabs,shrimps, lobsters and krills), wastes of the seafood process-ing industry[15,18,20,22–24]. Basically, the process consitsof deproteinization of the raw shell material with a diluteNaOH solution and decalcification with a dilute HCl solu-tion. To result in chitosan, the obtained chitin is subjectedto N-deacetylation by treatment with a 40–45% NaOH solu-tion, followed by purification procedures. Thus, productionand utilization of chitosan constitutes an economically at-tractive means of crustacean shell wastes disposal soughtworldwide.

Chitosan possesses distinct chemical and biologicalproperties[14–28a]. In its linear polyglucosamine chainsof high molecular weight, chitosan has reactive aminoand hydroxyl groups, amenable to chemical modifications[14,18,19,23]. Additionally, amino groups make chitosan acationic polyelectrolyte (pKa ≈ 6.5), one of the few found

130 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139

in nature. This basicity gives chitosan singular proper-ties: chitosan is soluble in aqueous acidic media at pH<

6.5 and when dissolved possesses high positive charge on–NH3

+ groups, it adheres to negatively charged surfaces,it aggregates with polyanionic compounds, and chelatesheavy metal ions. Both the solubility in acidic solutions andaggregation with polyanions impart chitosan with excel-lent gel-forming properties. Along with unique biologicalproperties that include biocompatibility, biodegradability toharmless products, nontoxicity, physiological inertness, re-markable affinity to proteins, hemostatic, fungistatic, antitu-moral and anticholesteremic properties, chitin and chitosan,as yet underutilized, offer an extraordinary potential in abroad spectrum of applications which are predicted to growrapidly once the standardized chitinous materials becomeavailable. Crucially, as bio- and biodegradable polymerschitin/chitosan materials are eco-friendly, safe for humansand the natural environment.

Increasingly over the last decade chitin- and chitosan-based materials have been examined and a number of po-tential products have been developed for areas such as[14,17,19,23,24,27,28b]wastewater treatment (removal ofheavy metal ions, flocculation/coagulation of dyes and pro-teins, membrane purification processes), the food industry(anticholesterol and fat binding, preservative, packaging ma-terial, animal feed additive), agriculture (seed and fertilizercoating, controlled agrochemical release), pulp and paperindustry (surface treatment, photographic paper), cosmeticsand toiletries (moisturizer, body creams, bath lotion).

But owing to the unparalleled biological properties,the most exciting uses of chitin/chitosan-based materi-als are those in the area of medicine and biotechnology[16,20–22,28a]. In medicine they may be employed as bac-teriostatic and fungistatic agents, drug delivery vehicles,drug controlled release systems, artificial cells, wound heal-ing ointments/dressings, haemodialysis membranes, contactlenses, artificial skin, surgical sutures and for tissue engi-neering. In biotechnology on the other hand, they may findapplication as chromatographic matrices, membranes formembrane separations, and notably as enzyme/cell immo-bilization supports.

As enzyme immobilization supports chitin- and chitosan-based materials are used in the form of powders, flakes andgels of different geometrical configurations. Chitin/chitosanpowders and flakes are available as commercial prod-ucts among others from Sigma-Aldrich and chitosan gelbeads (Chitopearl) from Fuji Spinning Co. Ltd. (Tokyo,Japan). Otherwise the chitinous supports are laboratory-manufactured. Preparation of chitosan gels is promoted bythe fact that chitosan dissolves readily in dilute solutionsof most organic acids, including formic, acetic, tartaricand citric acids, to form viscous solutions that precipitateupon an increase in pH and by formation of water-insolubleionotropic complexes with anionic polyelectrolytes. In thisway chitosan gels in the form of beads, membranes, coatings,capsules, fibres, hollow fibers and sponges can be manufac-

tured. Commonly, different follow-up treatments and modi-fications are applied to improve gel stability and durability.

The methods of chitosan gel preparation described in theliterature can be broadly divided into four groups: solventevaporation method, neutralization method, crosslinkingmethod and ionotropic gelation method[15,20,21,23–27].

3.1. Solvent evaporation method

The method is mainly used for the preparation of mem-branes and films, the latter being especially useful in prepar-ing minute enzymatically active surfaces deposited on tipsof electrodes. A solution of chitosan in organic acid is castonto a plate or an electrode tip and allowed to dry, if pos-sible at elevated temperature (ca. 65◦C). Upon drying themembrane/film is normally neutralized with a dilute NaOHsolution and crosslinked to avoid disintegration in solutionsof pH < 6.5. A crosslinking agent may also be mixed withthe initial chitosan solution before drying. Enzymes may beimmobilized on such prepared membranes either on theirsurfaces by adsorption, frequently followed by crosslinking(reticulation), or covalent binding, commonly preceded bychemical activation of the surface, or included into chitosansolution to achieve inclusion.

Spray drying is a variant of the solvent evaporationmethod allowing the preparation of beads smaller in sizethan those prepared with the other methods[44].

3.2. Neutralization method

If an acidic chitosan solution is mixed with alkali, an in-crease in pH results in precipitation of solid chitosan. Thismethod is exploited to produce chitosan precipitates, mem-branes, fibers, but foremost spherical beads of different sizesand porosities. These are obtained by adding a chitosansolution dropwise to a solution of NaOH, most frequentlyprepared in water-ethanol mixtures, where ethanol, beinga non-solvent for chitosan, facilitates the solidification ofchitosan beads. Following the preparation, the beads arecommonly subjected to crosslinking. Enzyme immobiliza-tion, similar to the solvent evaporation method, is achievedby binding onto the gel surface by adsorption, reticula-tion or covalent binding, or by inclusion if the enzyme isdissolved in the initial chitosan solution.

3.3. Crosslinking method

In this method an acidic chitosan solution is subjected tostraightforward crosslinking by mixing with a crosslinkingagent, which results in gelling. Gels obtained in bulk so-lution are later crushed into particles. To obtain gel mem-branes, the chitosan solution cast on a plate is immersedin a crosslinking bath, and to obtain beads the solution isadded dropwise therein. In the case of electrodes, crosslink-ing treatment is frequently done upon covering the tip of theelectrode with chitosan solution. Clearly, immobilization of

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Table 4Enzymes immobilized on chitin- and chitosan-based materials

Enzyme (EC number) Application Support (preparation method) Immobilization Reference

Acid phosphatase (3.1.3.2) F. Hydrophobic interaction chromatography; I. Mercapto-chitin powder I [29]Chitosan beads (b) III, IV [30,31]Chitosan precipitate (b) III [32]

Alanine dehydrogenase (1.4.1.1) E. Determination ofl-alanine (medicine) Chitosan beads III [33]

Alkaline phosphatase (3.1.3.1) F. Hydrophobic interaction chromatography Chitosan precipitate (b) III [32]C. Molecular cloning Chitosan beads (c) III [34]

Alkaline protease (3.4.21.62) B. Production of laundry detergents Chitin powder III (78%)a [35]Chitosan powder I (15%), III [35]

H. Ester and peptide synthesis; transesterification Chitosan beads I [36]

Alcohol dehydrogenase (1.1.1.1) I. Chitosan beads III (25%) [37]Chitosan membrane (a) III, IV [38]

Alcohol oxidase (1.1.3.13) E. Determination of ethanol Chitosan beads III [39]Aminoacylase (3.5.1.14) B. Production ofl-phenylalanine Chitosan-coated alginate beads (d) V (>100%) [40]

�-Amylase (3.2.1.1) A. Hydrolysis of starch for glucose syrup and Chitin powder III (38%)[41] [41,42]E. for BOD analysis in waters Chitosan beads I [43]F. Hydrophobic interaction chromatography Chitosan precipitate (b) III [32]

Chitosan microbeads (a) V [44]

�-Amylase (3.2.1.2) A. Production of high maltose syrup from starch Chitosan beads I [45]

�-l-Arabinofuranosidase (3.2.1.55) A. Aromatization of musts, alcoholic beverages andfruit juices

Chitosan powder I, II (3.2%)[47], III [46–48]Glyceryl-chitosan powder II [46]Chitosan particles (c) V [49]

Bromelain (3.4.22.32) I. Chitosan beads IV [50,51]Carbonic anhydrase (4.2.1.1) I. Chitosan-coated alginate beads (d) V [52]

Catalase (1.11.1.6) A. Removal of H2O2 from food Chitosan powder I, IV, II [53a,b]C. Treatment of hyperoxaluria Chitosan film (a) V [54]I. Chitosan membrane (a) III (4%) [55]

Chitosan-organosilane particles (c) I [56]Chitosan beads (c) V [57]

Cellulase (3.2.1.4) A. Decrease in viscosity of fruit/vegetable juices Chitin powder IV (15%) [58]F. Affinity chromatography Chitosan beads (b) I [59]

Chitosan solution protective additive [60]

Chitosanase (3.2.1.132) I. Chitin powder III [61]

�-Chymotrypsin (3.4.21.1) H. Ester and peptide synthesis Chitin film II [62]F. Preparation of trypsin-free chymotrypsin Chitosan beads I [36]

Chitosan-magnetite beads I [63]

Creatinine deaminase (3.5.4.21) D. Creatinine biosensor (medical diagnosis) Chitosan membrane (a) I, III [64]

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Cyclodextrin glycosyltransferase (2.4.1.19) I. Chitosan powder I (3.5%), IV(5.2%) [65]

Dextranase (3.2.1.11) C. Partial hydrolysis of dextran for preparation ofblood substitutes and B. of dentifrices

Chitin powder and colloidal chitin I, III [66]Chitosan powder I, III (63%) [66]

endo-1,4-�-Xylanase (3.2.1.8) C. Conversion of hemicelluloses (pulp industry) Chitosan powder I (24%) [67]Chitosan beads III (20%) [67]Chitosan-xanthan beads (d) V (180%)[70] [68–70]

Ficin (3.4.22.3) I. Chitosan beads IV [50]Galactose oxidase (1.1.3.9) D. Galactose biosensor Chitosan membrane (a) III [71a]

�-Galactosidase (3.2.1.22) A. Raffinose hydrolysis in beet molasses Chitin powder IV (67%) [71b,c]C. Blood group specificity; Fabry disease

�-Galactosidase (3.2.1.23) A. Hydrolysis of lactose (lactose-free dairy products) Chitin powder III [72,73]Chitosan powder III [74]Chitosan beads (b) III (100%)[75] [75,76]Chitosan beads I, III [77–79]Chitosan-polyphosphate beads (d) V [80]Chitosan precipitate (b) II [81]

Glucoamylase (3.2.1.3) A. Hydrolysis of starch (ethanol production) Chitin powder III [42]Chitosan magnetite beads (c) I [63]Chitosan powder I [82]Chitosan beads I, III [83]

Glucose oxidase (1.1.3.4) D. Glucose biosensors Chitin powder I [84]E. Determination of glucose �-Chitin membrane (coagulation) V [85,86]

Chitin film (coagulation) I [87]Chitosan beads III [88]Chitosan membrane (a) III [71a,89a,89b]Chitosan membrane (a, c, d) V [90–93a]Sol–gel/chitosan membrane (c) V [93b]Chitosan-organosilane particles (c) I [56,93c]Chitosan beads-liposomes III [94]

�-Glucosidase (3.2.1.20) A. Hydrolysis of maltose (food/feed additives) Chitosan beads III [95]

�-Glucosidase (3.2.1.21) A. Wine making and juice processing Chitosan powder III, II (29%)[98] [48,96–98]F. Hydrophobic interaction chromatography Chitosan particles (c) V [49,99]

Chitosan flakes III (60%), IV [100]Chitosan solution I (90%) [101]Chitosan beads (b) III, IV [31]Chitosan precipitate (b) III [32]Chitosan magnetite beads (c) I [63]

Glutamate dehydrogenase (1.4.1.2) E. Glutamate determination (food industry andmedicine)

Chitosan membrane (a) III [102]Succinyl-, glutaryl-, phtalyl-chitosanmembranes (a)

IV [102]

Glutamate oxidase (1.4.3.11) D. Glutamate biosensor Chitosan membrane (a) III [71a]

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Enzyme (EC number) Application Support (preparation method) Immobilization Reference

�-Glycosidase (3.2.1.group) A. Cellobiose hydrolysis for glucose production Chitosan powder II [103]Chitosan precipitate (b) II [104,105]

Horseradish peroxidase (1.11.1.7) D. H2O2 biosensor; E. determination of H2O2 Chitosan powder III, IV (62%)[106b] [106a,b]B. Oxidative polymerization of aniline Chitosan beads III [39,88,107]G. Removal of phenols from petroleum refinerywastewaters

Chitosan membrane (c) III [108]

E. Inhibition-based determination of Hg(II) Chitosan film (a) V [54,109,110]Chitosan solution Protective additive [111]Silica sol–gel chitosan film (c) I [112–114]Chitosan-carbon film (a) I [115]

Invertase (3.2.1.26) A. Hydrolysis of sucrose (production of invert sugar) Chitosan powder I (91%), III (44%), IV (70%)[116]Chitosan solution Protective additive [117]Chitosan microbeads (a) V [44]Chitosan-organosilane particles (c) I [56]Chitosan-magnetite beads (c) I [63]

Isoamylase (3.2.1.68) A. Hydrolysis of starch (glucose and maltose) Chitin powder III (46%) [118,119]

Laccase (1.10.3.2) B. Pulp and paper industry Chitosan precipitate (b) V, II (45%)[121] [120,121]G. Removal of phenols from effluents

Lactate oxidase (1.13.12.4) D. Lactate biosensor Chitosan-enzyme beads (d) V [122]Leucine dehydrogenase (1.4.1.9) E. Determination ofl-leucine (medicine) Chitosan beads III [33]

Limonoid glucosyltransferase (2.4.1.210) A. Debittering of citrus juice Chitosan powder III [123]Chitosan precipitate (b) III [123]

Lipase (3.1.1.3) H. Esterifications and transesterifications Chitosan flakes I (7.1%) [124]B. Hydrolysis of olive oil Chitosan beads I (14.7%)[124], IV [124–126a,c]

Chitosan beads IV+ II (91.5%) [126b]Chitosan-polyphosphate beads (d) V (42–50%) [127,128]Chitosan membrane (a) V, III (47%)[130] [129,130]Chitosan-PVA membrane (a) V [129]Chitosan-xanthan beads (d) V (90–99%) [131–133]

Lysozyme (3.2.1.17) F. Affinity membrane chromatography Microporous chitin membrane (a) I [134]A. Cheesemaking Chitosan powder I (10%) [135]

PHEMA-chitosan membranesmicroporous chitin membrane (a)

I [136–138]

Neutral proteinase (3.4.24.28) A. Hydrolysis of soybean protein Chitosan precipitate (b) II [139]Nucleoside phosphorylase (2.4.2.1) E. Determination of fish and shellfish freshness Chitosan beads III [140–142]5′-Nucleotidase (3.1.3.5) E. Determination of fish and shellfish freshness Chitosan beads III [140,142]Octopine dehydrogenase (1.5.1.11) E. Determination of shellfish freshness Chitosan beads III [143]Oxalate oxidase (1.2.3.4) C. Treatment of hyperoxaluria Chitosan powder II [53b]

Papain (3.4.22.2) A. Removal of “chill haze” in beers; I. Chitin powder II [144]B. Hydrolysis of collagen/keratin (cosmetics) Chitosan beads IV [50,145,146]

Chitosan precipitate (b) II (82%) [147]

Pectin lyase (4.2.2.10) A. Reduction of fruit/vegetable juices’ viscosity Chitin powder III (26%) [58]

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9Pectinase (3.2.1.15) C. Production of pectate oligosaccharides (inducers of flowering and

antibacterial agents)Chitosan beads I (15%) [148]

F. Hydrophobic interaction chromatography Chitosan precipitate (b) III [32]

Pepsin (3.4.23.1) I. Succinylated chitosan powder IV (80%) [149]Phospholipase A2 (3.1.1.4) C. Lowering plasma cholesterol level Chitosan beads IV (50%) [150]

Proteases (3.4.groups) A. Casein hydrolysate debittering; I. Chitin powder III [151]Chitin film II [62]Chitosan-xanthan beads (d) V [68,133]

Pullulanase (3.2.1.41) A. Hydrolysis of starch (glucose/maltose syrup) Chitin powder III [152]Chitosan-magnetite particles (c) IV [153]Chitosan powder I, III [152]Chitosan beads I [45]

Putrescine oxidase (1.4.3.10) E. Determination of meat freshness Chitosan beads III [154]

�-l-Rhamnopyranosidase (3.2.1.40) A. Aromatization of musts, alcoholic beverages and fruit juices Chitin powder II [155]Chitosan powder III, II [48,155]Chitosan particles (c) V [49]

Sulfite oxidase (1.8.3.1) D. Sulfite biosensor Chitosan-PHEMA membrane (b) I [156,157]

Tannase (3.1.1.20) A. Hydrolysis of tea tannins Chitin powder and colloidal chitin III, I [158]Chitosan precipitate (b) III [158]Chitosan-triphosphate beads (d) V [159]

Transglutaminase (2.3.2.13) A. Deamidation of food proteins Chitosan beads III [160]

Trypsin (3.4.21.4) F. Affinity purification Chitin flakes II, IV (67%) [161]A. Hydrolysis of proteins Chitosan-magnetite particles (c) I [162]

Tyrosinase (1.14.18.1) C. Production ofl-DOPA Chitin flakes III [163]G. Detection and removal of phenols Chitin powder I (95%) [164]

Chitosan flakes III [163,165]Chitosan beads (b) V (15%)[163], III [163,165]Chitosan-organosilane film (c) IV [166]Chitosan membrane (a, b) I [167,168]

Urease (3.5.1.5) C. Artificial kidney Chitosan-triphosphate beads (d) III (64%) [169]D. Urea biosensor Chitosan beads III (100%) [170]G. Treatment of fertilizer effluents Chitosan membrane (a) I, II, III (94%)[172] [171–173]A. Removal of urea from beverages and food Chitosan-PVA capsules (d) V [174]

Chitosan-PGMA precipitate (d) I (82%) [175]Chitosan-coated alginate beads (d) V [176]Chitosan-organosilane particles (c) I [56]

Uricase (1.7.3.3) E. Determination of uric acid (medicine) Chitosan membrane (a) IV [177]Xanthine oxidase (1.1.3.22) E. Determination of fish freshness Chitosan beads III [140–142]

�-Xylolidase (3.2.1.37) B. Production of lignocellulosic fibers Chitosan powder I (25%) [67]Chitosan beads III (33%) [67]

Applications are presented in nine cathegories: (A) food industry; (B) industries other than food; (C) medicine; (D) biosensors; (E) enzyme reactors for biosensing; (F) separation, purification andrecovery of enzymes; (G) environmental; (H) chemical synthesis; (I) immobilization studies. Support preparation methods are presented as: (a) solvent evaporation method; (b) neutralization method; (c)crosslinking method; (d) ionotropic gelation method. Commercial powders, flakes or gel beads are not marked. Immobilizations are presented as five techniques: (I) adsorption of enzyme on support; (II)adsorption of enzyme on support followed by cross-linking with glutaraldehyde (reticulation); (III) covalent binding of enzyme to glutaraldehyde-activated support; (IV) covalent binding of enzyme tosupport activated with agents other than glutaraldehyde; (V) gel inclusion.

a In brackets activity retention is given, if reported.

B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 135

enzymes on such prepared gels does not require chemicalactivation, as the crosslinker, normally a bifunctional agent,fullfils two functions, crosslinking and activation. The en-zyme may also be entrapped in the gel if mixed with chi-tosan prior to crosslinking.

Overwhelmingly, as a crosslinking and surface activatingagent glutaraldehyde is used. This is due to its reliabilityand ease of use, but more importantly, due to the availabil-ity of amino groups for the reaction with glutaraldehyde notonly on enzymes but also on chitosan. Other less frequentlyemployed difunctional agents include glyoxal[30,31,57],tris(hydroxymethyl)phosphine P(CH2OH)3 [38,100], hex-amethylenediamine[65,153], ethylenediamine[116], car-bodiimides[102,106b,126b,149], epichlorohydrin[129] andN-hydroxysuccinimide[50,51].

A comparatively newly developed method of chitosangelling is by use of sol–gel processes resulting in chitosan-organosilane hybrid gels. The method employs silylat-ing agents, such as (CH3O)3Si–R–NH2 [56], (CH3O)2-CH3Si–R–O–CO–CH=CH2 [113,166], (C2H5O)3Si–O–C2H5 [114], however, often regarded simply as crosslinkers.

3.4. Ionotropic gelation method (or coacervation)

By virtue of the attraction of oppositely-chargedmolecules, chitosan, owing to its cationic polyelectrolytenature, spontaneously forms water-insoluble complexeswith anionic polyelectrolytes[22,27,69]. The anionic poly-electrolytes used include alginate, carrageenan, xanthan,various polyphosphates and organic sulfates or enzymesthemselves[122]. The method is utilized chiefly for thepreparation of gel beads, which is achieved by adding ananionic polyelectrolyte solution dropwise into an acidicchitosan solution. Enzyme immobilization is achieved hereby preparing an enzyme-containing anionic polyelectrolytesolution prior to gelation. The enzyme is immobilized byinclusion in the interior of the beads/capsules.

An overview of enzymes immobilized on chitin- andchitosan-based materials, reported in the literature over thelast decade, is presented inTable 4. It implies that there con-tinues to be vivid interest in utilizing chitin-based materials,predominantly chitosan, as a promising enzyme immobi-lization support for a multiplicity of applications rangingfrom the wine, sugar and fish industries, through organiccontaminants removal from wastewaters to sophisticatedbiosensors for both in situ measurements of environmentalpollutants and metabolite control in artificial organs. Stud-ies like those summarized inTable 4can play a decisiverole in advancing this hitherto underutilized, renewablebiopolymer of great potential to the market of biomaterials.

Acknowledgments

This work was supported by the KBN grant no. PB7/T09A/048/20.

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