Enzima y Microbial Technology 35 (2004) 126-139

Aplicación de materiales chitin- y basados en

quitosano para inmovilizaciones enzimáticas: una
revisión
Barbara Krajewska*
Universidad Jagellónica, Facultad de Química, 30-060 Cracovia, Ingardena 3, Polonia
Recibido el 11 de septiembre de 2003; recibido en forma 24 de diciembre 2003 revisado; aceptan las 24 de
diciembre de de 2003

Resumen
Como materiales funcionales, quitina y quitosano ofrecen un conjunto único de características: biocompatibilidad, biodegradabilidad a
productos inocuos, no toxicidad, inercia fisiológica, propiedades antibacterianas, de metales pesados iones de quelación, propiedades de
formación de gel y la hidrofilia, y la afinidad notable a las proteínas. Debido a estas características, materiales chitin- y basados en
quitosano, como todavía subutilizado, se prevé que sean ampliamente explotados en un futuro próximo, especialmente en aplicaciones
ambientalmente benignos en los sistemas que trabajan en entornos biológicos, entre otros como soportes de inmovilización de enzimas.
Este trabajo es una revisión de la literatura sobre las enzimas inmovilizados sobre materiales chitin- y basados en quitosano, que cubren la
última década. Ciento cincuenta y ocho papeles en 63 enzimas inmovilizadas para la multiplicidad de aplicaciones que van desde el vino,
© 2004 Elsevier Inc. Todos los derechos reservados.
palabras clave: quitina; quitosano; la inmovilización de la enzima; aplicaciones; revisión

*
+ qué las enzimas?+
1. ¿Por Tel .: 48 12 6336377; Fax: 48 12 6340515.
Dirección de correo electrónico: krajewsk@chemia.uj.edu.pl (B.
Krajewska).
Si bien las metodologías convencionales de procesos
0141-0229 / $ - see front matter © 2004 Elsevier Inc. Todos los derechos
químicos se han desarrollado en las últimas décadas a un
reservados. doi: 10.1016 / j.enzmictec.2003.12.013
nivel permi- ing producción, separación y determinación
analítica de una enorme gama de productos sofisticados,
metodologías alternativas que no sólo son eficientes y
seguros, sino también ambientalmente benigna y en
recursos y ahorro de energía, se están buscando cada vez
más. Una de las estrategias más prometedoras para lograr
estos objetivos es la utilización de enzimas[1-5]. Las
enzimas exhiben una serie de características que hacen que
su uso ventajoso en comparación con catalizadores
químicos convencionales. La más importante de ellas son
un alto nivel de eficiencia catalítica, a menudo muy
superiores a los catalizadores químicos, y un alto grado de
especificidad que les permite discriminar no sólo entre las
reacciones sino también entre sustratos (especificidad
Strate sub-), partes similares de moléculas (regiospeci -
especifici-) y entre los isómeros ópticos
(estereoespecificidad). Estas especificidades garantizan que
la reacción catalizada no está perturbada por reacciones
secundarias, lo que resulta en la producción de uno quería
producto final, mientras que se elimina la producción de
indeseable subproductos. Esto proporciona rendimientos de
reacción sustancialmente más altas que reducen los costes
de material. Adicionalmente,
enzimas generalmente operan en condiciones suaves de
tem- Ature, presión y pH con velocidades de reacción del
orden de los alcanzados por catalizadores químicos en
condiciones más extremas. Esto hace que para el ahorro de
energía proceso sustanciales y los costes de fabricación
reducidos. Además, las enzimas prácticamente no
presentan problemas de eliminación, ya que, siendo en su
mayoría proteínas y péptidos, que son biodegradables y
fácilmente eliminado a partir de corrientes contaminadas.
Este conjunto único de características ventajosas de
enzimas como catalizadores ha sido explotado desde los
años 1960 y varios procesos catalizadas por enzimas se
han introducido con éxito para la industria, por ejemplo en
la producción de ciertos productos alimenticios, productos
farmacéuticos y productos agroquímicos, pero ahora
también cada vez más a la síntesis química orgánica.

2. ¿Por qué inmovilizar enzimas?

Además de las ventajas indiscutibles, existe una serie de

problemas prácticos en el uso de enzimas. A estos
pertenecen: el alto costo de aislamiento y purificación de
enzimas, la inestabilidad de sus estructuras una vez que
están lated iso- de sus entornos naturales, y su sensibilidad
tanto a las condiciones del proceso que no sean las
óptimas, normal- mente-a distancia estrecha, y para
rastrear niveles de sustancias que pueden actuar como
inhibidores. Los dos últimos resultado en vidas operativas
cortas enzimas. También, heterogéneo convencional a
diferencia
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 127

catalizadores químicos neos, la mayoría de las enzimas proceso de ensayo y error en una forma de asegurar la más alta
funcionan disuelven en agua en sistemas de catálisis retención posible de la actividad de la enzima, su estabilidad
homogénea, por lo que contaminan el producto y, como operacional y durabilidad.
regla no pueden ser Ered recu- en forma activa a partir de Ventajosa que sea, la inmovilización implica una serie de
mezclas de reacción para su reutilización. efectos empeorar el rendimiento de las enzimas [1-4,6,7]. En
Se han propuesto varios métodos para superar estas comparación con la enzima libre, más comúnmente
limitaciones, uno de los más exitosos enzima ser im-
movilización [1-6].La inmovilización se logra mediante la
fijación de las enzimas de o dentro de soportes sólidos,
como resultado de lo cual se obtienen sistemas de enzimas
inmovilizadas heterogéneos. Al imitar el modo natural de
ocurrencia en las células vivas, donde enzimas para la
mayoría de los casos están unidos a las membranas
celulares, los sistemas estabilizan la estructura de las
enzimas, por lo tanto, sus actividades. Por lo tanto, en
comparación con las enzimas libres en enzimas solución
inmovilizada son más robusto y más resistente a los
cambios ambientales. Más importante aún, la
heterogeneidad de los sistemas de enzimas inmovilizadas
permite una fácil recuperación tanto de la enzima y el
producto, la reutilización múltiple de las enzimas, la
operación continua de los procesos enzimáticos,
terminación rápida de reacciones y una mayor variedad de
diseños Actor biore-.
Las enzimas pueden inmovilizarse por una variedad de
métodos, que pueden ser ampliamente clasificados como
física, donde existen débiles interacciones in- entre el
apoyo y la enzima, y química, donde se forman enlaces
covalentes con la enzima [1-4,6,7]. Para los métodos físicos
pertenecen: (i) la contención de un Zyme en- dentro de un
reactor de membrana, (ii) de adsorción (física, iónico) en
una matriz insoluble en agua, (iii) la inclusión (o
atrapamiento gel), (iv ) microencapsulación con una
membrana sólida, (v) la microencapsulación con una
membrana de líquido, y
(Vi) formación de películas de Langmuir-Blodgett
enzimáticas. Los métodos de inmovilización química
incluyen: (i) covalente en- tachment a una matriz insoluble
en agua, (ii) reticular con el uso de un reactivo
multifuncional, de bajo peso molecular, y
(Iii) co-reticulación con otras sustancias neutras, por
ejemplo pro- teins. Numerosos otros métodos que son
combinaciones de los enumerados o original y específica
de un soporte o enzima dada se han ideado. Sin embargo,
hay un método único y apoyo es el mejor para todas las
enzimas y sus aplicaciones. Esto es debido a los
ampliamente diferentes carac- terísticas química y
composición de enzimas, las diferentes propiedades de los
sustratos y productos, y los diferentes usos a los que el
producto se puede aplicar. Además, todos los métodos
presentan ventajas y desventajas. La adsorción es simple,
barato y eficaz, pero con frecuencia reversible, la unión
covalente y la reticulación son eficaces y durable, pero es
caro y empeoramiento fácilmente el rendimiento de la
enzima, y en miem- brana reactor-confinment,
atrapamiento y problemas ciones microencapsula- de
difusión son inherentes. En consecuencia, por regla general
se encuentran las condiciones óptimas de inmovilización de
una enzima elegida y su aplicación empíricamente por un
 128 B. Krajewska / Enzyme and Microbial Technologycon
biológico 35 (2004) 126–139
un analito
y transducen en una respuesta
la enzima inmovilizada ha rebajado su actividad y la
constante de Michaelis aumentado. Estas alteraciones medible. Diferentes tipos de ERS transducción se han
son el resultado de cambios estructurales introducidos a empleado en biosensores potenciométricos a saber,
la enzima por el AP- manejó procedimiento de amperométrica, conductividad, termométrico, óptica y
inmovilización y de la creación de un microambiente en piezoeléctrico, la mayor parte de la investigación actual que
el que trabaja la enzima, diferente de la solución a está siendo colocado en los dos primeros. Enzimas para la
granel. Este último depende en gran medida teniendo mayoría de los casos se inmovilizan EI- Ther directamente
lugar la reacción, la naturaleza del soporte y en el diseño en la punta de trabajo de un transductor o en / sobre un
del reactor. Además, al ser sistemas de dos fases, los polímero
sistemas de enzimas inmovilizadas sufren de
limitaciones de transferencia de masa evitables in-,
produciendo efectos desfavorables sobre sus
rendimientos catalíticos en general. Estos, sin embargo,
pueden ser reducidos mediante la aplicación de diseños
de reactores apropiados.
Para la aplicación en un procedimiento comercial
todos los efectos ficiales y perjudiciales benefactores de
si se elige un catalizador químico o una enzima, y si se
utiliza una enzima libre o inmovilizada, han de ser
pesado teniendo en cuenta todos los aspectos
pertinentes, la salud y el medio ambiente incluidos , en
ad- DICIÓN a la viabilidad económica evidente. Hasta
la fecha, varios procesos basados en enzimas im-
movilizados han demostrado ser económico y se han
aplicado a mayor escala, principalmente en la industria
alimentaria, donde reemplazan a los procesos
catalizados por enzimas libres, y en la fabricación de
especialidades bien ningún producto químico y
farmacéutico , en particular cuando la síntesis o
resolución de enantiómeros asimétrica para producir
ópticamente puros productos están involucrados[1-5,8].
Una selección de los procesos de enzima inmovilizada
que se utilizan actualmente, en el der or- aproximada de
la escala decreciente de la fabricación, se da en Mesa
1. La escala de los procesos varía de aproximadamente 10 6 t
al año
para jarabe de maíz alto en fructosa, sin duda uno del
proceso basado en enzima inmovilizada comercialmente
importante más com-, a aproximadamente 102 t por año
para enantiopure L-DOPA [5].
Áreas de actuales y potenciales aplicaciones futuras
de im- movilizaron otros sistemas enzimáticos que
industrial (Mesa 1) incluir: síntesis orgánica escala de
laboratorio, y aplicaciones analíticas y médicas [1-5,7].
Después de haber sido demostrado ser capaces de
catalizar reacciones no sólo en soluciones acuosas, pero
también en medios orgánicos, enzimas ofrecen un gran
potencial para la síntesis orgánica sisting as- [9]. Ellos
pueden simplificar los procedimientos de iCal chem-
por reducir el número de etapas sintéticas, que pueden
mejorar la pureza de los productos, y más importante
aún, que pueden catalizar la síntesis de regio- y
estereoselectiva dar, compuestos de otra forma
imposibles de conseguir con las propiedades deseadas.
En aplicaciones analíticas enzimas inmovilizadas se
utilizan principalmente en biosensores [3,10-12] y en
menor medida, en las tiras de prueba diagnóstica. Los
biosensores se construyen mediante la integración de los
sistemas biológicos de detección, por ejemplo, la enzima
(s), con ERS transducción. Estos obtener una señal
química producida por la interacción del sistema
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 129

tabla 1
Algunas de las más importantes aplicaciones industriales de sistemas de enzimas
inmovilizadas [1-3,5] Enzima (CE número) SubstrateProduct

glucosa isomerasa (5.3.1.5) GlucoseFructose (maíz alto en fructosa jarabe)

TH-galactosidasa (3.2.1.23) LactoseGlucose y galactosa (sin lactosa de la leche y suero de leche)
lipasa (3.1.1.3) de mantequilla TriglyceridesCocoa sustitutos
nitrilohidratasa (4.2.1.84) AcrylonitrileAcrylamide
3-CyanopyridineNicotinamide
Adiponitrile5-Cyanovaleramide
aminoacilasa (3.5.1.14) D, L-AMINOÁCIDOS L-AMINO ácidos (Metionina, alanina, fenilalanina, triptófano, v a l i n a )
Raffinase (3.2.1.22) rafinosa galactosa y sacarosa (-Rafinosa libre soluciones)
invertasa (3.2.1.26) SucroseGlucose mezcla / fructosa (Invertir azúcar)
aspartato amoníaco-liasa (4.3.1.1) Amoníaco + fumárico ácido L-ASPÁRTICO ácido (usado para producción de sintético edulcorante
a s p a r t a m o ) termolisina (3.4.24.27) péptidos El aspartamo
glucoamilasa (3.2.1.3) Almidón D-GLUCOSA
Papaína (3.4.22.2) ProteinsRemoval de “neblina de enfriamiento” en las cervezas
hidantoinasa (3.5.2.2) D, L-AMINO a c i re h i d a n t o í n a s D, L-AMINO ácidos
amidasa de penicilina (3.5.1.11) Las penicilinas G y V6-aminopenicilánico ácido (precursor de penicilinas semisintéticas,
por ejemplo, ampicilina)
TH-tirosinasa (4.1.99.2) pirocatecol L-DOPA

membrana fuertemente envolviéndolo. En principio, debido
a la enzima de especificidad y sensibilidad biosensores se
pueden adaptar para casi cualquier analito diana, y estos
pueden ser ambos sustratos de enzimas e inhibidores de
enzimas. Ventajosamente, la deter- minación se realiza sin
una preparación especial de la muestra. Satisfacer la
demanda de dispositivos analíticos portátiles práctico,
rentable y, los biosensores basados en enzimas tienen un
enorme potencial como herramientas útiles en la medicina,
ambien- in situ y supervisión en tiempo real, de
bioprocesos y comida con- trol, y en el análisis biomédico
y farmacéutico . Su uso, sin embargo, por problemas de
fiabilidad como no bastante satisfactorio, se prevé que, una
vez llegado a ser ampliamente aceptado su almacenamiento
y estabilidades de funcionamiento se han mejorado.Mesa 2.
De éstos, sen- sores de glucosa son los que constituyen
aproximadamente más ampliamente estudiado 1/3 de la
literatura enzimáticos biosensores, los siguientes diez
sensores OC- Cupy otro tercio de la literatura y de los otros
sensores de la tercera restante [11]. Desde un punto de vista
práctico y comercial, cuatro de los sensores de la lista, es
decir, glucosa, lactato, urea y glutamato han sido
ampliamente utilizados [12].
Las aplicaciones médicas de las enzimas inmovilizadas
incluyen [1,4,13] el diagnóstico y tratamiento de
enfermedades, entre las terapias de reemplazo de la enzima,
así como células artificiales y órganos, y revestimiento de
materiales artificiales para una mejor biocompatibilidad.
Ofreciendo un gran potencial en esta área, la aplicación real
de las enzimas inmovilizadas hasta el momento ha sufrido
graves problemas de su toxicidad para el ganism or-
humano, hipoalergénico y las reacciones inmunológicas,
así como de su limitada estabilidad in vivo. Los ejemplos
de posibles usos médicos de sistemas de enzimas
inmovilizadas se enumeran enTabla 3.

Tabla 2
Algunas de las enzimas más frecuentemente estudiada para biosensores basados en enzimas [3,10-12]
Enzyme (número EC) sustrato Solicitud
La glucosa oxidasa (1.1.3.4) Glucosa Diagnóstico y tratamiento de la diabetes, la ciencia de los alimentos,
la biotecnología
peroxidasa de rábano picante (1.11.1.7) H2O2 Las aplicaciones biológicas e industriales, la inhibición de base
determinación de iones de metales pesados y pesticidas
Lactato oxidasa (1.13.12.4) lactato medicina deportiva, cuidados críticos, ciencia de los alimentos, la
biotecnología
La tirosinasa (1.14.18.1) Fenoles, polifenoles Determinación de los compuestos fenólicos en los alimentos, la
inhibición de base
determinación de pesticidas de carbamato
Glutamato oxidasa (1.4.3.11) Glutamato ciencia de los alimentos, la biotecnología
La ureasa (3.5.1.5) Urea diagnóstico médico, riñón artificial, monitoreo ambiental
alcohol deshidrogenasa (1.1.1.1) Etanol ciencia de los alimentos, la biotecnología
La acetilcolinesterasa (3.1.1.7) Acetilcolina, acetiltiocolina determinación basada en la inhibición de la organofosforados y
carbamatos
pesticidas
Colina oxidasa (1.1.3.17) La colina Enzima usada en conjunción con acetilcolinesterasa
La lactato deshidrogenasa (1.1.1.27) lactato medicina deportiva, cuidados críticos, ciencia de los alimentos, la
biotecnología
Colesterol oxidasa (1.1.3.6) Colesterol Aplicaciones medicas
128
Penicilinasa (3.5.2.6) B.penicilinas
Krajewska / Enzyme and Microbial Technology 35 (2004)
aplicaciones 126–139
farmacéuticas
Aliinasa (4.4.1.4) sulfóxidos cisteína Industria de la alimentación (Ajo, Cebollas y productos puerro
derivados)
 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139 131

Tabla 3 OH OH OH
Seleccionados usos médicos potenciales de las enzimas O O O
O O O
inmovilizadas [1,4,13] Enzima (CE número) Condición HO HO HO
NU NUEVA NH
asparaginasa (3.5.1.1) Leucemia EV HAMPSH C=
arginasa (3.5.3.1) Cáncer A IRE
C= O
La ureasa (3.5.1.5) riñón artificial, trastornos urémicos glucosa HA
MP La CH3
oxidasa (1.1.3.4) Artificial páncreas O
SHI quitina
CH3
RE
C=
O
CH3
deshidratasa carbonato (4.2.1.1) pulmones
OH OH OH
+ Catalasa (1.11.1.6) artificiales O O O
catalasa (1.11.1.6) acatalasemia O O O
glucoamilasa (3.2.1.3) de almacenamiento de glucógeno HO NH2 HO NH2
enfermedad NUEVA
HO
deshidrogenasa de glucosa- Glucosa-6-fosfato deshidrogenasa
HAMPSHIRE2
6-fosfato (1.1.1.49) El quitosano
La xantina oxidasa (1.1.3.22) Lesch-Nyhan enfermedad
fenilalanina amonio liasa fenilcetonuria
OH OH OH
(4.3.1.5) O O O
urato oxidasa (1.7.3.3) La hiperuricemia O O O
heparinasa (4.2.2.7), la terapia extracorpórea procedimientos HO HO HO
OH OH OH
Celulosa
3. ¿Por qué inmovilizar enzimas en chitin- y
materiales a base de quitosano? constituyente principal de las conchas de los crustáceos, los
exoesqueletos de insectos y las paredes celulares de los
Las propiedades de las enzimas inmovilizadas se rigen hongos donde proporciona resistencia y estabilidad, se estima
por las propiedades tanto de la enzima y el material de que la quitina que sintetizarse y degradado en la biosfera en la
soporte [4,6]. La interacción entre los dos se presta una gran cantidad de al menos 10 Gt cada año. Químicamente, la
propiedades cinéticas que pueden ser decisivo para su quitina se compone de
aplicación práctica, y por lo tanto, un puerto SUP- þ (1 → 4) vinculado unidades de 2-acetamido-2-desoxi-TH-d-
juiciosamente elegido puede mejorar significativamente el glucosa
rendimiento fun- cional del sistema inmovilizado enzima
inmovilizada específica físico-químicas y. Aunque se
reconoce que no hay apoyo universal para todas las
enzimas y sus aplicaciones, una serie de características
deseables debe ser común a cualquier material considerado
para enzimas bilizing inmovilidad. Estos incluyen: alta
afinidad a las proteínas, la disponibilidad de grupos
funcionales reactivos para las reacciones directas con
enzimas y para las modificaciones químicas, dad hidrófilo-,
estabilidad mecánica y rigidez, regenerabilidad, y facilidad
de preparación en diferentes configuraciones geométricas
que proporcionan el sistema con la permeabilidad y el área
superficial traje- capaz para una biotransformación elegido.
Es comprensible que, para alimentos, productos
farmacéuticos, aplicaciones médicas y agrícolas, no
toxicidad y la biocompatibilidad de los materiales también
son obligatorios. Por otra parte, para responder a la
creciente de salud pública y la conciencia ambiental, los
materiales deben ser biodegradables, y para demostrar
económico, barato.
De las muchas portadoras que han sido considerados y
estu- IED para la inmovilización de enzimas, orgánicos o
inorgánicos, naturales o sintéticos, quitina y quitosano son
de interés ya que ofrecen la mayor parte de las
características anteriores.
La quitina y el quitosano son polyaminosaccharides
naturales [14-28],quitina es uno de la mayoría de los
recursos del mundo plenti- ful, renovables orgánicos. Un
 128 B. Krajewska
Fig. 1. Estructura de la quitina, quitosano / Enzyme and Microbial Technology 35 (2004) 126–139
y celulosa.

(o N-acetil-D-glucosamina) [14], la formación de un

polímero lineal de cadena larga (Higo. 1).Es insoluble
en la mayoría de disolventes. Chitosan, el derivado
principal de la quitina, se obtiene mediante la N-
desacetilación en un grado variable que se caracteriza
por el grado de desacetilación, y es en consecuencia un
mer copoly- de N-acetil-D-glucosamina y D-
glucosamina. La quitina y el quitosano pueden ser
considerados químicamente como análogos de celulosa,
en los que el hidroxilo en el carbono-2 se ha sustituido
por grupos acetamido y amino, respectivamente. Tosan
Chi es insoluble en agua, pero la presencia de grupos
amino hace que sea soluble en soluciones ácidas por
debajo de un pH de aproximadamente 6,5. Es importante
tener en cuenta que la quitina y el quitosano no son
entidades químicas individuales, sino que varían en la
composición dependiendo del proceso de origen y la
fabricación. Chitosan puede ser definida como la quitina
desacetilada suficientemente para formar sales de amina
solubles,
Comercialmente, la quitina y el quitosano se obtienen
en una rel- tivamente bajo coste a partir de conchas de
los moluscos (principalmente cangrejos, gambas,
langostas y krills), residuos de los productos del mar
proceso- industria ing [15,18,20,22-24]. Básicamente, el
proceso consiste de desproteinización del material de la
cáscara en bruto con una solución diluida de NaOH y
descalcificación con una solución de HCl diluido. Para
resultar en quitosano, la quitina obtenido se somete a N-
desacetilación, por tratamiento con una solu- ción NaOH
40-45%, seguido de procedimientos de purificación. Por
lo tanto, la producción y la utilización de quitosano
constituye un medio de tracción económicamente At-
crustáceo de la eliminación de residuos de cáscara
buscado en todo el mundo.
Chitosan posee propiedades biológicas distintas
química y [14-28a]. En sus cadenas poliglucosamina
lineales de alto peso molecular, el quitosano tiene
grupos amino e hidroxilo reactivos, susceptibles de
modificaciones químicas [14,18,19,23]. Además, los
grupos amino hacen quitosano una
polielectrolito catiónico (pKa ≈ 6.5), uno de los pocos
conocer
130 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139

en naturaleza. Esta basicidad da propie- dades singulares de way chitosan gels in the form of beads, membranes,
quitosano: quitosano es soluble en medio ácido acuoso a coatings, capsules, fibres, hollow fibers and sponges can be
pH < manufac-
6,5 y cuando se disuelve posee carga positiva alta en
NH3+ grupos, se adhiere a superficies cargadas
negativamente, agregados con compuestos polianiónicos, y
quelatos iones de metales pesados. Tanto la solubilidad en
soluciones ácidas y la agregación con polianiones imparten
quitosano con exce- propiedades de formación de gel
Cuaresma. Junto con propiedades biológicas únicas que
incluyen biocompatibilidad, biodegradabilidad inofensivos
productos, no toxicidad, inercia fisiológica, afinidad
marcable re- a las proteínas, hemostático, fungistático,
propiedades morales y Anticolesterémicos antitu-, quitina y
quitosano, como todavía subutilizado, ofrecer un
extraordinario potencial en un amplio espectro de
aplicaciones que se prevé que crezca rápidamente una vez
que los materiales de quitina estandarizados estén
disponibles. Fundamentalmente, como polímeros bio y
biodegradables materiales de quitosano de quitina / son
ecológicos, seguro para los seres humanos y el medio
ambiente natural.
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
128 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139
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
130 B. Krajewska / Enzyme and Microbial Technology 35 (2004) 126–139
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. D e t e r m i n a t i o n o f 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]
a-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]
þ-Amylase (3.2.1.2) A. Production of high maltose syrup from starch Chitosan beads I [45]
a-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 H2O2 from food Chitosan powder I, IV, II [53a,b]
C. Treatment of hyperoxaluria Chitosan film (a) V [54]
I. Chitosan membrane (a) III (4%) [55]
Chitosan-organosilane particles (c) I [56]
Chitosan beads (c) V [57]
Cellulase (3.2.1.4) A. Decrease in viscosity of fruit/vegetable juices Chitin powder IV (15%) [58]
F. Affinity chromatography Chitosan beads (b) I [59]
Chitosan solution protective additive [60]
Chitosanase (3.2.1.132) I. Chitin powder III [61]
a-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-þ-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]
a-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

þ-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 þ-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]
a-Glucosidase (3.2.1.20) A. Hydrolysis of maltose (food/feed additives) Chitosan beads III [95]
þ-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
þ-Glycosidase (3.2.1.group) A. Cellobiose hydrolysis for glucose production Chitosan powder II [103]
Chitosan precipitate (b) II [104,105]
Horseradish peroxidase (1.11.1.7) D. H2O2 biosensor; E. determination of H2O2 Chitosan powder III, IV (62%) [106b] [106a,b]
B. Oxidative polymerization of aniline Chitosan beads III [39,88,107]
G. Removal of phenols from petroleum 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. D e t e r m i n a t i o n o f 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]
5J -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]
Pectinase (3.2.1.15) C. Production of pectate oligosaccharides (inducers of flowering and Chitosan beads I (15%) [148]

134
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

a-l-Rhamnopyranosidase A. Aromatization of musts, alcoholic beverages and fruit juices Chitin powder II [155]
(3.2.1.40)
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]
þ-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(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.
135
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