Enzyme and Microbial Technology 35 (2004) 126–139

Application of chitin- and chitosan-based materials

for 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, 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? enzymes generally operate at mild conditions of temper-

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 (stereospecificity).
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).
0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2003.12.013
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-
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139
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 membrane, 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
127
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 pharmaceuticals, 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 106 t per year
for high-fructose corn syrup, arguably one of the most com-
mercially important immobilized enzyme-based process, to
about 102 t 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]
Enzyme (EC number) Substrate Product

Glucose isomerase (5.3.1.5) Glucose Fructose (high-fructose corn syrup)

␤-Galactosidase (3.2.1.23) Lactose
Lipase (3.1.1.3) Triglycerides
Nitrile hydratase (4.2.1.84) Acrylonitrile
3-Cyanopyridine
Adiponitrile
Aminoacylase (3.5.1.14) d,l-Aminoacids
Raffinase (3.2.1.22) Raffinose
Invertase (3.2.1.26) Sucrose
Aspartate ammonia-lyase (4.3.1.1) Ammonia + fumaric acid
Thermolysin (3.4.24.27) Peptides
Glucoamylase (3.2.1.3) Starch
Papain (3.4.22.2) Proteins
Hydantoinase (3.5.2.2) d,l-Amino acid hydantoins
Penicillin amidase (3.5.1.11) Penicillins G and V
␤-Tyrosinase (4.1.99.2) Pyrocatechol
Glucose and galactose (lactose-free milk and whey)
Cocoa butter substitutes
Acrylamide
Nicotinamide
5-Cyanovaleramide
l-Amino acids (methionine, alanine, phenylalanine, tryptophan, valine)
Galactose and sucrose (raffinose-free solutions)
Glucose/fructose mixture (invert sugar)
l-Aspartic acid (used for production of synthetic sweetener aspartame)
Aspartame
d-Glucose
Removal of “chill haze” in beers
d,l-Amino acids
6-Aminopenicillanic acid (precursor of semi-synthetic penicillins,
e.g. ampicillin)
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
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.

Table 2
Some of the most frequently studied enzymes for enzyme-based biosensors [3,10–12]
Enzyme (EC number) Substrate Application

Glucose oxidase (1.1.3.4)
Horseradish peroxidase (1.11.1.7)
Lactate oxidase (1.13.12.4)
Tyrosinase (1.14.18.1)
Glutamate oxidase (1.4.3.11)
Urease (3.5.1.5)
Alcohol dehydrogenase (1.1.1.1)
Acetylcholinesterase (3.1.1.7)
Choline oxidase (1.1.3.17)
Lactate dehydrogenase (1.1.1.27)
Cholesterol oxidase (1.1.3.6)
Penicillinase (3.5.2.6)
Alliinase (4.4.1.4)
Glucose Diagnosis and treatment of diabetes, food science, biotechnology
H 2 O2 Biological and industrial applications, inhibition-based
determination of heavy metal ions and pesticides
Lactate Sports medicine, critical care, food science, biotechnology
Phenols, polyphenols Determination of phenolic compounds in foods, inhibition-based
determination of carbamate pesticides
Glutamate Food science, biotechnology
Urea Medical diagnosis, artificial kidney, environmental monitoring
Ethanol Food science, biotechnology
Acetylcholine, acetylthiocholine Inhibition-based determination of organophosphorus and carbamate
pesticides
Choline Enzyme used in conjunction with acetycholinesterase
lactate Sports medicine, critical care, food science, biotechnology
Cholesterol Medical applications
Penicillins Pharmaceutical applications
Cysteine sulfoxides Food industry (garlic-, onions- and leek-derived products)
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 129

Table 3 OH OH OH
Selected potential medical uses of immobilized enzymes [1,4,13] O O O
O O O
Enzyme (EC number) Condition HO HO HO
NH NH NH
Asparaginase (3.5.1.1) Leukemia C=O C=O C=O
Arginase (3.5.3.1) Cancer CH3 CH3 CH3
Urease (3.5.1.5) Artificial kidney, uraemic disorders Chitin
Glucose oxidase (1.1.3.4) Artificial pancreas
Carbonate dehydratase (4.2.1.1) Artificial lungs
OH OH OH
+ catalase (1.11.1.6) O O O
Catalase (1.11.1.6) Acatalasemia O O O
Glucoamylase (3.2.1.3) Glycogen storage disease HO HO HO
Glucose-6-phosphate Glucose-6-phosphate dehydrogenase NH2 NH2 NH2
dehydrogenase (1.1.1.49) deficiency Chitosan
Xanthine oxidase (1.1.3.22) Lesch–Nyhan disease
Phenylalanine ammonia lyase Phenylketonuria
(4.3.1.5) OH OH OH
O O O
Urate oxidase (1.7.3.3) Hyperuricemia O O O
Heparinase (4.2.2.7) Extracorporeal therapy procedures HO HO HO
OH OH OH
Cellulose
3. Why immobilize enzymes on chitin- and
chitosan-based materials? Fig. 1. Structure of chitin, chitosan and cellulose.

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 rigidity, regenerability, 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
(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 (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 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/dressings, 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 covering the tip of the
electrode with chitosan solution. Clearly, immobilization of
Table 4
Enzymes 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 of l-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]

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

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 of l-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 and Chitosan powder I, II (3.2%) [47], III [46–48]
fruit juices 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 H2 O2 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]

131
 132
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 of Chitin powder and colloidal chitin I, III [66]
blood substitutes and B. of dentifrices 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

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

␤-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 and Chitosan membrane (a) III [102]
medicine) Succinyl-, glutaryl-, phtalyl-chitosan IV [102]
membranes (a)
Glutamate oxidase (1.4.3.11) D. Glutamate biosensor Chitosan membrane (a) III [71a]
Table 4 (Continued )
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. H2 O2 biosensor; E. determination of H2 O2 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 refinery Chitosan membrane (c) III [108]
wastewaters
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]

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

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 of l-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 membranes I [136–138]
microporous chitin membrane (a)
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]

133
Pectin lyase (4.2.2.10) A. Reduction of fruit/vegetable juices’ viscosity Chitin powder III (26%) [58]
 134
Pectinase (3.2.1.15) C. Production of pectate oligosaccharides (inducers of flowering and Chitosan beads I (15%) [148]
antibacterial agents)
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]

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

␣-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 of l-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 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.
a In brackets activity retention is given, if reported.
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139
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(CH2 OH)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 (CH3 O)3 Si–R–NH2 [56], (CH3 O)2 -
CH3 Si–R–O–CO–CH=CH2 [113,166], (C2 H5 O)3 Si–O–
C2 H5 [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.
135
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