Application of chitin and chitosanbased materials for enzyme immobilizations: a review

Enzyme and Microbial Technology 35 2004 126–139Application of chitin- and chitosan-based materials for enzyme immobilizations: a review Jagiellonian University, Faculty of Chemistry, 30-

Enzyme and Microbial Technology 35 (2004) 126–139

Application of chitin- and chitosan-based materials

for enzyme immobilizations: a review

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, and remarkable affinity to proteins Owing to these characteristics, chitin- and chitosan-based materials, as yet underutilized, are predicted to be widely exploited in the near future especially in environmentally benign applications in systems working in biological environments, among others as enzyme immobilization supports This paper is a review of the literature on enzymes immobilized on chitin- and chitosan-based materials, covering the last decade One hundred fifty-eight papers on 63 immobilized enzymes for multiplicity of applications ranging from wine, sugar and fish industry, through organic compounds removal from wastewaters to sophisticated biosensors for both in situ measurements 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 processes

have been developed in the past decades to a level

allow-ing production, separation and analytical determination of

an enormous range of sophisticated products, alternative

methodologies that are not only efficient and safe but also

environmentally benign and resource- and energy-saving,

are being increasingly sought One of the most promising

strategies to achieve these goals is the utilization of enzymes

[1–5] Enzymes exhibit a number of features that make their

use advantageous as compared to conventional chemical

catalysts Foremost among them are a high level of catalytic

efficiency, often far superior to chemical catalysts, and a

high degree of specificity that allows them to discriminate

not only between reactions but also between substrates

(sub-strate specificity), similar parts of molecules

(regiospeci-ficity) and between optical isomers (stereospeci(regiospeci-ficity).

These specificities warrant that the catalyzed reaction is not

perturbated by side-reactions, resulting in the production

of one wanted end-product, whereas production of

undesir-able by-products is eliminated This provides substantially

higher 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 of those achieved by chemical catalysts at more extreme condi-tions This makes for substantial process energy savings and reduced manufacturing costs Also, enzymes practically do not present disposal problems since, being mostly proteins and peptides, they are biodegradable and easily removed from contaminated streams This unique set of advantageous features of enzymes as catalysts has been exploited since the 1960s and several enzyme-catalyzed processes have been successfully introduced to industry, e.g in the production

of certain foodstuffs, pharmaceuticals and agrochemicals, but now also increasingly to organic chemical synthesis.

2 Why immobilize enzymes?

In addition to the unquestionable advantages, there exists

a number of practical problems in the use of enzymes To these belong: the high cost of isolation and purification of enzymes, the instability of their structures once they are iso-lated from their natural environments, and their sensitivity both to process conditions other than the optimal ones, nor-mally narrow-ranged, and to trace levels of substances that can act as inhibitors The latter two result in enzymes’ short operational 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 dissolved

in water in homogeneous catalysis systems, which is why

they 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 these

limitations, one of the most successful being enzyme

im-mobilization [1–6] Immobilization is achieved by fixing

enzymes to or within solid supports, as a result of which

heterogeneous immobilized enzyme systems are obtained.

By mimicking the natural mode of occurence in living cells,

where enzymes for the most cases are attached to cellular

membranes, the systems stabilize the structure of enzymes,

hence their activities Thus, as compared to free enzymes

in solution immobilized enzymes are more robust and more

resistant to environmental changes More importantly, the

heterogeneity of the immobilized enzyme systems allows

easy recovery of both enzyme and product, multiple reuse

of 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 (or

gel entrapment), (iv) microencapsulation with a solid

mem-brane, (v) microencapsulation with a liquid memmem-brane, and

(vi) formation of enzymatic Langmuir-Blodgett films The

chemical immobilization methods include: (i) covalent

at-tachment to a water-insoluble matrix, (ii) crosslinking with

use 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 of

the ones listed or original and specific of a given support

or enzyme have been devised However, no single method

and 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 of

substrates and products, and the different uses to which the

product can be applied Besides, all of the methods present

advantages and drawbacks Adsorption is simple, cheap and

effective but frequently reversible, covalent attachment and

crosslinking are effective and durable, but expensive and

easily worsening the enzyme performance, and in

mem-brane reactor-confinment, entrapment and

microencapsula-tions diffusional problems are inherent Consequently, as a

rule the optimal immobilization conditions for a chosen

en-zyme and its application are found empirically by a process

of trial and error in a way to ensure the highest possible

retention of activity of the enzyme, its operational stability

and durability.

Advantageous though it is, the immobilization involves a

number 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 the Michaelis constant increased These alterations result from structural changes introduced to the enzyme by the ap-plied immobilization procedure and from the creation of

a microenvironment in which the enzyme works, different from the bulk solution The latter is strongly dependent on the reaction taking place, the nature of the support and on the design of the reactor Furthermore, being two phase systems, the immobilized enzyme systems suffer from in-evitable mass transfer limitations, producing unfavourable effects on their overall catalytic performances These, however, may be reduced by applying appropriate reactor designs.

For the implementation in a commercial process all bene-ficial and detrimental effects of whether a chemical catalyst

or an enzyme is chosen, and whether a free or immobilized enzyme is used, have to be weighed taking into account all relevant aspects, health and environmental included, in ad-dition to obvious economical viability To date, several im-mobilized enzyme-based processes have proved economic and have been implemented on a larger scale, mainly in the food industry, where they replace free enzyme-catalyzed processes, and in the manufacture of fine speciality chem-icals and pharmaceutchem-icals, particularly where asymmetric synthesis or resolution of enantiomers to produce optically pure products are involved [1–5,8] A selection of currently used immobilized-enzyme processes, in the approximate or-der of the decreasing scale of manufacture, is given in Table

1 The scale of the processes ranges from about 106t per year for high-fructose corn syrup, arguably one of the most com-mercially important immobilized enzyme-based process, to about 102t per year for enantiopure l-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 analytical and medical applications [1–5,7] Having been shown to be able to catalyze reactions not only in aqueous solutions but also 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 synthesis giving, otherwise unobtainable compounds with the desired properties.

In analytical applications immobilized enzymes are used chiefly in biosensors [3,10–12] and to a lesser extent, in diag-nostic test strips Biosensors are constructed by integrating biological 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 transduce

it into a measurable response Different kinds of transduc-ers have been employed in biosensors, viz potentiometric, amperometric, conductometric, thermometric, optical and piezo-electric, most of the current research being placed on the 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 1

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

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)

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)

Aspartate ammonia-lyase (4.3.1.1) Ammonia+ fumaric acid l-Aspartic acid (used for production of synthetic sweetener aspartame)

Hydantoinase (3.5.2.2) d,l-Amino acid hydantoins d,l-Amino acids

Penicillin 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 tailored

for nearly any target analyte, and these can be both enzyme

substrates and enzyme inhibitors Advantageously, their

de-termination is performed without special preparation of the

sample Meeting the demand for practical, cost-effective and

portable analytical devices, enzyme-based biosensors have

enormous 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 Their

use, impaired as yet by not quite satisfactory reliability, is

predicted to become widely accepted once their storage and

operational stabilities have been improved The most

exten-sively studied enzymes for the application in enzyme-based

biosensors are presented in Table 2 Of these, glucose

sen-sors are the most widely studied constituting ca 1/3 of the

Table 2

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

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

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

determination of carbamate pesticides

Acetylcholinesterase (3.1.1.7) Acetylcholine, acetylthiocholine Inhibition-based determination of organophosphorus and carbamate

pesticides Choline oxidase (1.1.3.17) Choline Enzyme used in conjunction with acetycholinesterase

Lactate dehydrogenase (1.1.1.27) lactate Sports medicine, critical care, food science, biotechnology

Alliinase (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 the remaining 1/3 [11] From a practical and commercial point

of 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 those enzyme replacement therapies, as well as artificial cells and organs, and coating of artificial materials for better bio-compatibility Offering a great potential in this area, real application of immobilized enzymes has as yet suffered from serious problems from their toxicity to the human or-ganism, allergenic and immunological reactions as well as from their limited stability in vivo Examples of potential medical uses of immobilized enzyme systems are listed in Table 3.

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

Table 3

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

Enzyme (EC number) Condition

Asparaginase (3.5.1.1) Leukemia

Urease (3.5.1.5) Artificial kidney, uraemic disorders

Glucose oxidase (1.1.3.4) Artificial pancreas

Carbonate dehydratase (4.2.1.1)

+ catalase (1.11.1.6)

Artificial lungs

Catalase (1.11.1.6) Acatalasemia

Glucoamylase (3.2.1.3) Glycogen storage disease

Glucose-6-phosphate

dehydrogenase (1.1.1.49)

Glucose-6-phosphate dehydrogenase deficiency

Xanthine oxidase (1.1.3.22) Lesch–Nyhan disease

Phenylalanine ammonia lyase

(4.3.1.5)

Phenylketonuria

Urate oxidase (1.7.3.3) Hyperuricemia

Heparinase (4.2.2.7) Extracorporeal therapy procedures

3 Why immobilize enzymes on chitin- and

chitosan-based materials?

The properties of immobilized enzymes are governed by

the properties of both the enzyme and the support material

[4,6] The interaction between the two lends an immobilized

enzyme specific physico-chemical and kinetic properties that

may 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 is

recognized that there is no universal support for all enzymes

and their applications, a number of desirable characteristics

should be common to any material considered for

immo-bilizing enzymes These include: high affinity to proteins,

availability of reactive functional groups for direct reactions

with enzymes and for chemical modifications,

hydrophilic-ity, mechanical stability and rigidhydrophilic-ity, regenerabilhydrophilic-ity, and ease

of preparation in different geometrical configurations that

provide the system with permeability and surface area

suit-able for a chosen biotransformation Understandably, for

food, pharmaceutical, medical and agricultural applications,

nontoxicity and biocompatibility of the materials are also

required Furthermore, to respond to the growing public

health and environmental awareness, the materials should be

biodegradable, and to prove economical, inexpensive.

Of the many carriers that have been considered and

stud-ied for immobilizing enzymes, organic or inorganic, natural

or synthetic, chitin and chitosan are of interest in that they

offer 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 constituent

of the shells of crustaceans, the exoskeletons of insects

and the cell walls of fungi where it provides strength

and stability, chitin is estimated to be synthesized and

degraded in the biosphere in the vast amount of at

least 10 Gt each year Chemically, chitin is composed of

␤(1 → 4) linked 2-acetamido-2-deoxy-␤-d-glucose units

O OH HO O OH

O OH HO O

O OH HO O OH

O OH HO O

O OH HO O OH

O OH HO O

NH C=O

CH3

NH C=O

CH3

NH C=O

CH3

Chitin

Chitosan

Cellulose

O O

O

Fig 1 Structure of chitin, chitosan and cellulose

(or N-acetyl- d-glucosamine) [14], forming a long chain linear polymer (Fig 1) It is insoluble in most solvents Chitosan, the principal derivative of chitin, is obtained by

N-deacetylation to a varying extent that is characterized by

the degree of deacetylation, and is consequently a

copoly-mer of N-acetyl- d-glucosamine and d-glucosamine Chitin and chitosan can be chemically considered as analogues

of cellulose, in which the hydroxyl at carbon-2 has been replaced by acetamido and amino groups, respectively Chi-tosan is insoluble in water, but the presence of amino groups renders it soluble in acidic solutions below pH about 6.5 It

is important to note that chitin and chitosan are not single chemical entities, but vary in composition depending on the origin and manufacture process Chitosan can be defined as chitin sufficiently deacetylated to form soluble amine salts, the degree of deacetylation necessary to obtain a soluble product 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 consits

of deproteinization of the raw shell material with a dilute NaOH solution and decalcification with a dilute HCl solu-tion To result in chitosan, the obtained chitin is subjected

to N-deacetylation by treatment with a 40–45% NaOH

solu-tion, followed by purification procedures Thus, production and utilization of chitosan constitutes an economically at-tractive means of crustacean shell wastes disposal sought worldwide.

Chitosan possesses distinct chemical and biological properties [14–28a] In its linear polyglucosamine chains

of high molecular weight, chitosan has reactive amino and hydroxyl groups, amenable to chemical modifications [14,18,19,23] Additionally, amino groups make chitosan a

cationic polyelectrolyte (pK ≈ 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 chelates

heavy metal ions Both the solubility in acidic solutions and

aggregation with polyanions impart chitosan with

excel-lent gel-forming properties Along with unique biological

properties that include biocompatibility, biodegradability to

harmless 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 a

broad spectrum of applications which are predicted to grow

rapidly once the standardized chitinous materials become

available Crucially, as bio- and biodegradable polymers

chitin/chitosan materials are eco-friendly, safe for humans

and 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 of

heavy 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 fertilizer

coating, controlled agrochemical release), pulp and paper

industry (surface treatment, photographic paper), cosmetics

and 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/dressheal-ings, haemodialysis membranes, contact

lenses, artificial skin, surgical sutures and for tissue

engi-neering In biotechnology on the other hand, they may find

application as chromatographic matrices, membranes for

membrane 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 and

gels of different geometrical configurations Chitin/chitosan

powders and flakes are available as commercial

prod-ucts among others from Sigma-Aldrich and chitosan gel

beads (Chitopearl) from Fuji Spinning Co Ltd (Tokyo,

Japan) Otherwise the chitinous supports are

laboratory-manufactured Preparation of chitosan gels is promoted by

the fact that chitosan dissolves readily in dilute solutions

of most organic acids, including formic, acetic, tartaric

and citric acids, to form viscous solutions that precipitate

upon an increase in pH and by formation of water-insoluble

ionotropic complexes with anionic polyelectrolytes In this

way 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 the literature can be broadly divided into four groups: solvent evaporation method, neutralization method, crosslinking method 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 tips

of electrodes A solution of chitosan in organic acid is cast onto a plate or an electrode tip and allowed to dry, if pos-sible at elevated temperature (ca 65◦C) Upon drying the membrane/film is normally neutralized with a dilute NaOH solution and crosslinked to avoid disintegration in solutions

of pH < 6.5 A crosslinking agent may also be mixed with

the initial chitosan solution before drying Enzymes may be immobilized on such prepared membranes either on their surfaces by adsorption, frequently followed by crosslinking (reticulation), or covalent binding, commonly preceded by chemical activation of the surface, or included into chitosan solution to achieve inclusion.

Spray drying is a variant of the solvent evaporation method allowing the preparation of beads smaller in size than 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 This method is exploited to produce chitosan precipitates, mem-branes, fibers, but foremost spherical beads of different sizes and porosities These are obtained by adding a chitosan solution dropwise to a solution of NaOH, most frequently prepared in water-ethanol mixtures, where ethanol, being

a non-solvent for chitosan, facilitates the solidification of chitosan beads Following the preparation, the beads are commonly subjected to crosslinking Enzyme immobiliza-tion, similar to the solvent evaporation method, is achieved

by binding onto the gel surface by adsorption, reticula-tion or covalent binding, or by inclusion if the enzyme is dissolved in the initial chitosan solution.

3.3 Crosslinking method

In this method an acidic chitosan solution is subjected to straightforward crosslinking by mixing with a crosslinking agent, 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 immersed

in a crosslinking bath, and to obtain beads the solution is added dropwise therein In the case of electrodes, crosslink-ing treatment is frequently done upon covercrosslink-ing the tip of the electrode with chitosan solution Clearly, immobilization of

Table 4

Enzymes immobilized on chitin- and chitosan-based materials

␣-l-Arabinofuranosidase (3.2.1.55) A Aromatization of musts, alcoholic beverages and

fruit juices

blood substitutes and B of dentifrices

C Blood group specificity; Fabry disease

medicine)

Succinyl-, glutaryl-, phtalyl-chitosan membranes (a)

Table 4 (Continued )

G Removal of phenols from petroleum refinery wastewaters

G Removal of phenols from effluents

PHEMA-chitosan membranes microporous chitin membrane (a)

antibacterial agents)

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 and recovery 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 to support activated with agents other than glutaraldehyde; (V) gel inclusion

aIn 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 chemical

activation, 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 activating

agent glutaraldehyde is used This is due to its reliability

and ease of use, but more importantly, due to the

availabil-ity of amino groups for the reaction with glutaraldehyde not

only on enzymes but also on chitosan Other less frequently

employed 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] and

N-hydroxysuccinimide [50,51].

A comparatively newly developed method of chitosan

gelling 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-charged

molecules, chitosan, owing to its cationic polyelectrolyte

nature, spontaneously forms water-insoluble complexes

with anionic polyelectrolytes [22,27,69] The anionic

poly-electrolytes used include alginate, carrageenan, xanthan,

various polyphosphates and organic sulfates or enzymes

themselves [122] The method is utilized chiefly for the

preparation of gel beads, which is achieved by adding an

anionic polyelectrolyte solution dropwise into an acidic

chitosan solution Enzyme immobilization is achieved here

by preparing an enzyme-containing anionic polyelectrolyte

solution prior to gelation The enzyme is immobilized by

inclusion in the interior of the beads/capsules.

An overview of enzymes immobilized on chitin- and

chitosan-based materials, reported in the literature over the

last decade, is presented in Table 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 ranging

from the wine, sugar and fish industries, through organic

contaminants removal from wastewaters to sophisticated

biosensors for both in situ measurements of environmental

pollutants and metabolite control in artificial organs

Stud-ies like those summarized in Table 4 can play a decisive

role in advancing this hitherto underutilized, renewable

biopolymer of great potential to the market of biomaterials.

Acknowledgments

This work was supported by the KBN grant no PB

7/T09A/048/20.

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