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

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?
While conventional methodologies of chemical processes
have been developed in the past decades to a level allowing 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 (substrate specificity), similar parts of molecules (regiospecificity) 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 undesirable 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: (B. Krajewska).

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

enzymes generally operate at mild conditions of temperature, pressure and pH with reaction rates of the order of
those achieved by chemical catalysts at more extreme conditions. 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 isolated from their natural environments, and their sensitivity
both to process conditions other than the optimal ones, normally 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 recovered 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 immobilization [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 bioreactor designs.
Enzymes may be immobilized by a variety of methods,
which may be broadly classified as physical, where weak interactions 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 enzyme within a membrane reactor, (ii) adsorption (physical, ionic) on a water-insoluble matrix, (iii) inclusion (or

gel entrapment), (iv) microencapsulation with a solid membrane, (v) microencapsulation with a liquid membrane, and
(vi) formation of enzymatic Langmuir-Blodgett films. The
chemical immobilization methods include: (i) covalent attachment 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. proteins. 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 characteristics 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 membrane reactor-confinment, entrapment and microencapsulations diffusional problems are inherent. Consequently, as a
rule the optimal immobilization conditions for a chosen enzyme 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 applied 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 inevitable 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 beneficial 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 addition to obvious economical viability. To date, several immobilized 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 chemicals 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 order 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 commercially important immobilized enzyme-based process, to
about 102 t per year for enantiopure l-DOPA [5].
Areas of present and potential future applications of immobilized 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 assisting organic synthesis [9]. They can simplify the chemical procedures by reducing the number of synthetic steps,
they can enhance the purity of the products, and most importantly, 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 diagnostic test strips. Biosensors are constructed by integrating
biological sensing systems, e.g. enzyme(s), with transducers. These obtain a chemical signal produced by the interaction of the biological system with an analyte and transduce

it into a measurable response. Different kinds of transducers 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 either directly on a transducer’s working tip or in/on a polymer


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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)
␤-Galactosidase (3.2.1.23)
Lipase (3.1.1.3)
Nitrile hydratase (4.2.1.84)

Aminoacylase (3.5.1.14)
Raffinase (3.2.1.22)
Invertase (3.2.1.26)
Aspartate ammonia-lyase (4.3.1.1)
Thermolysin (3.4.24.27)
Glucoamylase (3.2.1.3)
Papain (3.4.22.2)

Hydantoinase (3.5.2.2)
Penicillin amidase (3.5.1.11)

Glucose
Lactose
Triglycerides
Acrylonitrile
3-Cyanopyridine
Adiponitrile
d,l-Aminoacids
Raffinose
Sucrose
Ammonia + fumaric acid
Peptides
Starch
Proteins
d,l-Amino acid hydantoins
Penicillins G and V

␤-Tyrosinase (4.1.99.2)

Pyrocatechol

Fructose (high-fructose corn syrup)
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 enzyme specificity and sensitivity biosensors can be tailored
for nearly any target analyte, and these can be both enzyme
substrates and enzyme inhibitors. Advantageously, their determination 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, environmental in situ and real time monitoring, bioprocess and food control, 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 extensively studied enzymes for the application in enzyme-based
biosensors are presented in Table 2. Of these, glucose sensors are the most widely studied constituting ca. 1/3 of the

enzyme-biosensors literature, the subsequent ten sensors occupy 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 biocompatibility. 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 organism, 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)

Glucose
H 2 O2

Lactate oxidase (1.13.12.4)
Tyrosinase (1.14.18.1)

Lactate
Phenols, polyphenols

Glutamate oxidase (1.4.3.11)
Urease (3.5.1.5)
Alcohol dehydrogenase (1.1.1.1)
Acetylcholinesterase (3.1.1.7)

Glutamate
Urea
Ethanol
Acetylcholine, acetylthiocholine

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)

Choline
lactate
Cholesterol
Penicillins
Cysteine sulfoxides

Diagnosis and treatment of diabetes, food science, biotechnology
Biological and industrial applications, inhibition-based
determination of heavy metal ions and pesticides
Sports medicine, critical care, food science, biotechnology
Determination of phenolic compounds in foods, inhibition-based
determination of carbamate pesticides
Food science, biotechnology
Medical diagnosis, artificial kidney, environmental monitoring
Food science, biotechnology
Inhibition-based determination of organophosphorus and carbamate
pesticides
Enzyme used in conjunction with acetycholinesterase

Sports medicine, critical care, food science, biotechnology
Medical applications
Pharmaceutical applications
Food industry (garlic-, onions- and leek-derived products)


B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139
Table 3
Selected potential medical uses of immobilized enzymes [1,4,13]
Enzyme (EC number)

Condition

Asparaginase (3.5.1.1)
Arginase (3.5.3.1)
Urease (3.5.1.5)
Glucose oxidase (1.1.3.4)
Carbonate dehydratase (4.2.1.1)
+ catalase (1.11.1.6)
Catalase (1.11.1.6)
Glucoamylase (3.2.1.3)
Glucose-6-phosphate
dehydrogenase (1.1.1.49)
Xanthine oxidase (1.1.3.22)
Phenylalanine ammonia lyase
(4.3.1.5)
Urate oxidase (1.7.3.3)
Heparinase (4.2.2.7)

Leukemia

Cancer
Artificial kidney, uraemic disorders
Artificial pancreas
Artificial lungs
Acatalasemia
Glycogen storage disease
Glucose-6-phosphate dehydrogenase
deficiency
Lesch–Nyhan disease
Phenylketonuria
Hyperuricemia
Extracorporeal therapy procedures

OH
HO

O
NH
C=O
CH3

OH
HO

NH2

OH
HO

O

O
OH

129

OH
O
HO

Chitin

O
NH
C=O
CH3

OH
O
HO

O
NH2

OH
O
HO

OH

O

OH
O
HO

OH

O

O

NH2

OH
O
HO

O

NH
C=O
CH3

Chitosan

O
HO

O

O

O

OH

Cellulose

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 support judiciously chosen can significantly enhance the operational 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 immobilizing enzymes. These include: high affinity to proteins,
availability of reactive functional groups for direct reactions
with enzymes and for chemical modifications, hydrophilicity, mechanical stability and rigidity, regenerability, and ease
of preparation in different geometrical configurations that
provide the system with permeability and surface area suitable 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 studied 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 plentiful, 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

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 copolymer 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. Chitosan 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 relatively low cost from shells of shellfish (mainly crabs,
shrimps, lobsters and krills), wastes of the seafood processing 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 solution. To result in chitosan, the obtained chitin is subjected
to N-deacetylation by treatment with a 40–45% NaOH solution, followed by purification procedures. Thus, production
and utilization of chitosan constitutes an economically attractive 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


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

in nature. This basicity gives chitosan singular properties: 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 excellent gel-forming properties. Along with unique biological
properties that include biocompatibility, biodegradability to
harmless products, nontoxicity, physiological inertness, remarkable affinity to proteins, hemostatic, fungistatic, antitumoral 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 chitosanbased materials have been examined and a number of potential 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 proteins, membrane purification processes), the food industry
(anticholesterol and fat binding, preservative, packaging material, 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 materials are those in the area of medicine and biotechnology
[16,20–22,28a]. In medicine they may be employed as bacteriostatic and fungistatic agents, drug delivery vehicles,
drug controlled release systems, artificial cells, wound healing ointments/dressings, haemodialysis membranes, contact
lenses, artificial skin, surgical sutures and for tissue engineering. In biotechnology on the other hand, they may find
application as chromatographic matrices, membranes for
membrane separations, and notably as enzyme/cell immobilization supports.
As enzyme immobilization supports chitin- and chitosanbased materials are used in the form of powders, flakes and
gels of different geometrical configurations. Chitin/chitosan
powders and flakes are available as commercial products among others from Sigma-Aldrich and chitosan gel
beads (Chitopearl) from Fuji Spinning Co. Ltd. (Tokyo,
Japan). Otherwise the chitinous supports are laboratorymanufactured. 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 modifications 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 membranes and films, the latter being especially useful in preparing 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 possible 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 increase in pH results in precipitation of solid chitosan. This
method is exploited to produce chitosan precipitates, membranes, 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 immobilization, similar to the solvent evaporation method, is achieved
by binding onto the gel surface by adsorption, reticulation 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 solution are later crushed into particles. To obtain gel membranes, 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, crosslinking 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
Application

Support (preparation method)

Immobilization

Reference

Acid phosphatase (3.1.3.2)

F. Hydrophobic interaction chromatography; I.

Mercapto-chitin powder
Chitosan beads (b)
Chitosan precipitate (b)

I
III, IV
III

[29]
[30,31]
[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
C. Molecular cloning

Chitosan precipitate (b)
Chitosan beads (c)

III
III

[32]
[34]

Alkaline protease (3.4.21.62)

B. Production of laundry detergents

Chitin powder
Chitosan powder

III (78%)a
I (15%), III

[35]
[35]

H. Ester and peptide synthesis; transesterification

Chitosan beads

I

[36]

Alcohol dehydrogenase (1.1.1.1)

I.

Chitosan beads
Chitosan membrane (a)

III (25%)
III, IV

[37]

[38]

Alcohol oxidase (1.1.3.13)
Aminoacylase (3.5.1.14)

E. Determination of ethanol
B. Production of l-phenylalanine

Chitosan beads
Chitosan-coated alginate beads (d)

III
V (>100%)

[39]
[40]

␣-Amylase (3.2.1.1)

A. Hydrolysis of starch for glucose syrup and
E. for BOD analysis in waters
F. Hydrophobic interaction chromatography

Chitin powder
Chitosan beads
Chitosan precipitate (b)
Chitosan microbeads (a)

III (38%) [41]
I

III
V

[41,42]
[43]
[32]
[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
fruit juices

Bromelain (3.4.22.32)
Carbonic anhydrase (4.2.1.1)

I.
I.

Chitosan powder

Glyceryl-chitosan powder
Chitosan particles (c)
Chitosan beads
Chitosan-coated alginate beads (d)

I, II (3.2%) [47], III
II
V
IV
V

[46–48]
[46]
[49]
[50,51]
[52]

Catalase (1.11.1.6)

A. Removal of H2 O2 from food
C. Treatment of hyperoxaluria
I.

Chitosan powder
Chitosan film (a)
Chitosan membrane (a)
Chitosan-organosilane particles (c)
Chitosan beads (c)

I, IV, II

V
III (4%)
I
V

[53a,b]
[54]
[55]
[56]
[57]

Cellulase (3.2.1.4)

A. Decrease in viscosity of fruit/vegetable juices
F. Affinity chromatography

Chitin powder
Chitosan beads (b)
Chitosan solution

IV (15%)
I
protective additive

[58]
[59]
[60]

Chitosanase (3.2.1.132)

I.

Chitin powder

III

[61]

␣-Chymotrypsin (3.4.21.1)

H. Ester and peptide synthesis
F. Preparation of trypsin-free chymotrypsin

Chitin film
Chitosan beads
Chitosan-magnetite beads

II
I
I

[62]
[36]
[63]

Creatinine deaminase (3.5.4.21)

D. Creatinine biosensor (medical diagnosis)

Chitosan membrane (a)

I, III

[64]

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

Enzyme (EC number)

131


132

I.

Chitosan powder

I (3.5%), IV(5.2%)

[65]

Dextranase (3.2.1.11)

C. Partial hydrolysis of dextran for preparation of
blood substitutes and B. of dentifrices

Chitin powder and colloidal chitin
Chitosan powder

I, III
I, III (63%)

[66]
[66]

endo-1,4-␤-Xylanase (3.2.1.8)

C. Conversion of hemicelluloses (pulp industry)

Chitosan powder
Chitosan beads
Chitosan-xanthan beads (d)

I (24%)
III (20%)
V (180%) [70]

[67]
[67]
[68–70]

Ficin (3.4.22.3)
Galactose oxidase (1.1.3.9)

I.
D. Galactose biosensor

Chitosan beads
Chitosan membrane (a)

IV
III

[50]
[71a]

␣-Galactosidase (3.2.1.22)

A. Raffinose hydrolysis in beet molasses
C. Blood group specificity; Fabry disease

Chitin powder

IV (67%)

[71b,c]

␤-Galactosidase (3.2.1.23)

A. Hydrolysis of lactose (lactose-free dairy products)

Chitin powder
Chitosan powder
Chitosan beads (b)
Chitosan beads
Chitosan-polyphosphate beads (d)
Chitosan precipitate (b)

III

III
III (100%) [75]
I, III
V
II

[72,73]
[74]
[75,76]
[77–79]
[80]
[81]

Glucoamylase (3.2.1.3)

A. Hydrolysis of starch (ethanol production)

Chitin powder
Chitosan magnetite beads (c)
Chitosan powder
Chitosan beads

III
I
I
I, III

[42]
[63]
[82]

[83]

Glucose oxidase (1.1.3.4)

D. Glucose biosensors
E. Determination of glucose

Chitin powder
␤-Chitin membrane (coagulation)
Chitin film (coagulation)
Chitosan beads
Chitosan membrane (a)
Chitosan membrane (a, c, d)
Sol–gel/chitosan membrane (c)
Chitosan-organosilane particles (c)
Chitosan beads-liposomes

I
V
I
III
III
V
V
I
III

[84]
[85,86]
[87]

[88]
[71a,89a,89b]
[90–93a]
[93b]
[56,93c]
[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
F. Hydrophobic interaction chromatography

Chitosan
Chitosan
Chitosan
Chitosan
Chitosan
Chitosan
Chitosan

III, II (29%) [98]
V
III (60%), IV
I (90%)
III, IV
III
I

[48,96–98]
[49,99]
[100]
[101]
[31]
[32]
[63]

Glutamate dehydrogenase (1.4.1.2)

E. Glutamate determination (food industry and
medicine)

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

III
IV

[102]
[102]

Glutamate oxidase (1.4.3.11)

D. Glutamate biosensor

Chitosan membrane (a)

III

[71a]

powder
particles (c)
flakes
solution
beads (b)
precipitate (b)
magnetite beads (c)

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

Cyclodextrin glycosyltransferase (2.4.1.19)


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
Chitosan precipitate (b)

II
II

[103]
[104,105]

Horseradish peroxidase (1.11.1.7)

D. H2 O2 biosensor; E. determination of H2 O2
B. Oxidative polymerization of aniline
G. Removal of phenols from petroleum refinery
wastewaters
E. Inhibition-based determination of Hg(II)

Chitosan powder
Chitosan beads
Chitosan membrane (c)

III, IV (62%) [106b]
III
III

[106a,b]
[39,88,107]
[108]

Chitosan film (a)
Chitosan solution
Silica sol–gel chitosan film (c)
Chitosan-carbon film (a)

V
Protective additive
I
I

[54,109,110]
[111]
[112–114]
[115]

Chitosan powder
Chitosan solution
Chitosan microbeads (a)
Chitosan-organosilane particles (c)
Chitosan-magnetite beads (c)

I (91%), III (44%), IV (70%)

Protective additive
V
I
I

[116]
[117]
[44]
[56]
[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
G. Removal of phenols from effluents

Chitosan precipitate (b)

V, II (45%) [121]

[120,121]

Lactate oxidase (1.13.12.4)
Leucine dehydrogenase (1.4.1.9)

D. Lactate biosensor
E. Determination of l-leucine (medicine)

Chitosan-enzyme beads (d)
Chitosan beads

V
III

[122]
[33]

Limonoid glucosyltransferase (2.4.1.210)

A. Debittering of citrus juice

Chitosan powder
Chitosan precipitate (b)

III
III

[123]
[123]

Lipase (3.1.1.3)

H. Esterifications and transesterifications
B. Hydrolysis of olive oil

Chitosan flakes
Chitosan beads
Chitosan beads
Chitosan-polyphosphate beads (d)
Chitosan membrane (a)
Chitosan-PVA membrane (a)
Chitosan-xanthan beads (d)

I (7.1%)
I (14.7%) [124], IV
IV + II (91.5%)
V (42–50%)
V, III (47%) [130]
V
V (90–99%)

[124]
[124–126a,c]
[126b]
[127,128]
[129,130]
[129]
[131–133]

Lysozyme (3.2.1.17)

F. Affinity membrane chromatography
A. Cheesemaking

Microporous chitin membrane (a)
Chitosan powder
PHEMA-chitosan membranes
microporous chitin membrane (a)

I
I (10%)
I

[134]
[135]
[136–138]

Neutral proteinase (3.4.24.28)
Nucleoside phosphorylase (2.4.2.1)
5 -Nucleotidase (3.1.3.5)
Octopine dehydrogenase (1.5.1.11)
Oxalate oxidase (1.2.3.4)

A. Hydrolysis of soybean protein
E. Determination of fish and shellfish freshness
E. Determination of fish and shellfish freshness
E. Determination of shellfish freshness
C. Treatment of hyperoxaluria

Chitosan

Chitosan
Chitosan
Chitosan
Chitosan

II
III
III
III
II

[139]
[140–142]
[140,142]
[143]
[53b]

Papain (3.4.22.2)

A. Removal of “chill haze” in beers; I.
B. Hydrolysis of collagen/keratin (cosmetics)

Chitin powder
Chitosan beads
Chitosan precipitate (b)

II
IV
II (82%)

[144]
[50,145,146]
[147]

Pectin lyase (4.2.2.10)

A. Reduction of fruit/vegetable juices’ viscosity

Chitin powder

III (26%)

[58]

precipitate (b)
beads
beads
beads
powder

133

A. Hydrolysis of sucrose (production of invert sugar)

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

Invertase (3.2.1.26)


Chitosan beads

I (15%)

[148]

Chitosan precipitate (b)

III

[32]

Pepsin (3.4.23.1)
Phospholipase A2 (3.1.1.4)

I.
C. Lowering plasma cholesterol level

Succinylated chitosan powder
Chitosan beads

IV (80%)
IV (50%)

[149]
[150]

Proteases (3.4.groups)

A. Casein hydrolysate debittering; I.

Chitin powder
Chitin film
Chitosan-xanthan beads (d)

III
II
V

[151]
[62]
[68,133]

Pullulanase (3.2.1.41)

A. Hydrolysis of starch (glucose/maltose syrup)

Chitin powder
Chitosan-magnetite particles (c)
Chitosan powder
Chitosan beads

III
IV
I, III
I

[152]
[153]
[152]
[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
Chitosan powder
Chitosan particles (c)

II
III, II
V

[155]
[48,155]
[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
Chitosan precipitate (b)
Chitosan-triphosphate beads (d)

III, I
III
V

[158]
[158]
[159]

Transglutaminase (2.3.2.13)

A. Deamidation of food proteins

Chitosan beads

III

[160]

Trypsin (3.4.21.4)

F. Affinity purification
A. Hydrolysis of proteins

Chitin flakes
Chitosan-magnetite particles (c)

II, IV (67%)
I

[161]
[162]

Tyrosinase (1.14.18.1)

C. Production of l-DOPA
G. Detection and removal of phenols

Chitin flakes
Chitin powder
Chitosan flakes
Chitosan beads (b)
Chitosan-organosilane film (c)
Chitosan membrane (a, b)

III

I (95%)
III
V (15%) [163], III
IV
I

[163]
[164]
[163,165]
[163,165]
[166]
[167,168]

Urease (3.5.1.5)

C.
D.
G.
A.

Chitosan-triphosphate beads (d)
Chitosan beads
Chitosan membrane (a)
Chitosan-PVA capsules (d)
Chitosan-PGMA precipitate (d)
Chitosan-coated alginate beads (d)
Chitosan-organosilane particles (c)

III (64%)
III (100%)

I, II, III (94%) [172]
V
I (82%)
V
I

[169]
[170]
[171–173]
[174]
[175]
[176]
[56]

Uricase (1.7.3.3)
Xanthine oxidase (1.1.3.22)

E. Determination of uric acid (medicine)
E. Determination of fish freshness

Chitosan membrane (a)
Chitosan beads

IV
III

[177]
[140–142]

␤-Xylolidase (3.2.1.37)

B. Production of lignocellulosic fibers

Chitosan powder
Chitosan beads

I (25%)
III (33%)

[67]
[67]

Artificial kidney
Urea biosensor
Treatment of fertilizer effluents
Removal of urea from beverages and food

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

C. Production of pectate oligosaccharides (inducers of flowering and
antibacterial agents)
F. Hydrophobic interaction chromatography

134

Pectinase (3.2.1.15)


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 enzyme may also be entrapped in the gel if mixed with chitosan 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 availability 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], hexamethylenediamine [65,153], ethylenediamine [116], carbodiimides [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 chitosanorganosilane hybrid gels. The method employs silylating 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 polyelectrolytes 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 continues to be vivid interest in utilizing chitin-based materials,
predominantly chitosan, as a promising enzyme immobilization 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. Studies 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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