Revista Mexicana de Fı́sica S58 (2) 39–43 DICIEMBRE 2012
Immobilization of mannanase on magnetic chitosan microspheres
L.V. Zuluagab, O.H Giraldoa, and C.E. Orregoa,∗
aDepartment of Physics and Chemistry, National Universty of Colombia,
Cra 27 # 64-60, Manizales,
bDepartment of Chemical Engineering, National University of Colombia,
Cra 27 # 64-60, Manizales.
Recibido el 25 de junio de 2010; aceptado el 28 de marzo de 2011
Chitosan has been widely investigated for the enzyme immobilization. In this study, magnetic chitosan microspheres (MCM) were synthe-
sized with a ferrofluid, using ammonium bicarbonate to make insoluble chitosan; these microspheres were used for enzyme immobilization.
Acetic acid (1%w/v) solution was used as solvent of chitosan dispersion (2%w/v), this dispersion was let react with a ferrofluid (2%v/v) for
2 hours with continuous stirring, then the enzyme was added, and after a while all solution was dried in a spray dryer obtaining magnetic chi-
tosan microspheres with immobilized enzyme, characterization of microspheres were performed with SEM/ESEM micrographs confirmed
spherical morphology. Energy-Dispersive X-Ray Spectroscopy (EDX) was used for the detection of iron in the samples. Additional the
magnetization profile were obtained.
Mannanase was immobilized on magnetic chitosan microspheres by entrapment and cross-linking with glutaraldehyde. The immobilization
conditions were investigated achieving the conservation of catalytic activity. The catalytic behavior of the immobilized mannanase was
acceptable as compared with the same characteristics of the free enzyme. The magnetic characteristic of these materials allows easy removal
of enzyme after use.
Keywords: Magnetic chitosan microspheres; immobilization; entrapment; cross linking.
El quitosano ha sido investigado ampliamente para la inmovilización de enzimas. En este estudio, se sintetizaron microesferas magnéticas de
quitosano (MCM) usando un ferrofluido; para insolubilizar el quitosano se utilizó bicarbonato de amonio; estas microesferas fueron usadas
para la inmovilizacíon de enzimas. Una solución de acido aćetico (1%w/v) fue usada como solvente para la dispersión de quitosano (2%w/v),
esta dispersión se dejo reaccionar con un ferrofluido (2%v/v) por 2 horas con agitación continua, se agregó la enzima y despúes de un tiempo
toda la dispersión fue secada en un secador por aspersión obteniendo microesferas magnéticas de quitosano con enzima inmovilizada, la car-
acterizacíon de las microesferas fue realizada con micrografı́as SEM/ESEM confirmando su morfologı́a esf́erica. Espectroscopia de energı́a
dispersa de rayos X (EDX) fue usada para la detección del hierro en las muestras. Adicionalmente se obtuvo el perfil de magnetización.
Una mananasa fue inmovilizada en las microesferas magnéticas de quitosano por atrapamiento y entrecruzamiento con glutaraldehido. Las
condiciones de la inmovilización fueron investigadas logrando la conservación de la actividad catalı́tica. El comportamiento catalı́tico de
la mananasa inmovilizada fue aceptable en comparación con la enzima libre en iguales condiciones. Las caracterı́sticas magńeticas de este
material permiten remover fácilmente la enzima después de uso para utilizarla nuevamente.
Descriptores: Microesferas magńeticas de quitosano; inmovilización; atrapamiento; entrecruzamiento.
PACS: 75.75.-c; 75.75.Cd; 75.60.Ej; 75.70.Cn; 75.47.Lx
One of the major sources of renewable organic matter is the
hemicellulose, a complex group of polymers. In the cell wall
of plants the mannan is one of the major constituent and the
enzymes that degrade it have found applications in the phar-
maceutical, food, pulp and paper industries .
In the food industry, mannan degrading enzymes may
be used for the maceration of fruit and vegetable materials
and clarification of juices and wines , in the extraction of
vegetable oils from leguminous seeds, the viscosity reduc-
tion in extracts during the manufacture of instant coffee ,
improvement in the consistency of beer, and biopulping of
wood, especially softwood and to improve the gelling prop-
erties of galactomannans to be used as food thickeners [1,3].
It’s also used as a food supplement for animals (chickens and
pigs), allowing greater digestion and assimilation of nutri-
ents [4,5]. β-Mannanases were introduced into the deter-
gent market as agents against reappearing stains during laun-
Many enzymes used in the food industry are quite ex-
pensive, but when they are insolubilized by coupling them to
an adequate matrix, the resulting biocatalyst may be reused
several times, thus lowering the costs , immobilization
also allows better reactions control and permits the design
of bioreactors that can be easily incorporated into a continu-
ous processing line . The properties of immobilized en-
zymes are governed by the properties of both the enzyme
and the support material [7,9]. One important attribute for
using a support in the food industry is to be inert and non-
toxic. One of the options was chitin and chitosan which offer
a unique set of characteristics: biocompatibility, biodegrad-
ability to harmless products, non-toxic, physiological inert-
ness, antibacterial properties, heavy metal ions chelation, gel
40 L.V. ZULUAGA, O.H GIRALDO, AND C.E. ORREGO
forming properties and hydrophilicity, and remarkable affin-
ity to proteins [9,10]. Chitosan with low degree of acetyla-
tion is highly soluble in water so, the formation of a poly-
cation with ammonium carbonate make the biopolymer in-
soluble and give the possibility of obtain microspheres for
In recent years, magnetic carrier technology has showed
significant attractive for the preparation of immobilized en-
zymes. Chitosan can be used as a base material for magnetic
carriers . Magnetic chitosan is a great support for en-
zyme immobilization because is very easy to remove the im-
mobilized enzyme of the reaction medium with the help of a
The aim of this work was to prepare soluble
polyelectrolyte-magnetic nanoparticles complexes in solu-
tions suitable for spray-drying to obtain, after drying, water
insoluble polyelectrolyte-magnetic micro-spheres (PMM).
Using two different protocols, PMM were employed to man-
ufacture immobilized mannanase that were characterized by
measurements of their catalytic and magnetic properties.
2. Materials and methods
Reagents and materials
Ferric chloride (FeCl3.6H2O), ferrous chloride
(FeCl2.4H2O), ammonia and kerosene were all chemical
grade and chitosan flakes (high molecular weight 602 kDa,
degree of deacetylation 76.5%) were obtained from Sigma
Chemical Co. (St. Louis, MO, United States), Ro-
halase GMP (an enzyme preparation that contains man-
nanase as main activity ) with a nominal specific activity
of 1.000.000 MNU g−1 solid and containing 44% protein
based on the Bradford protein assay were obtained from AB
Enzymes (Darmstadt, Germany), citrus pectin (methoxy con-
tent 60 %) was purchased from Cp Kelko (Sau Paulo, Brazil)
and oleic acid from Carlo Erba (Milan, Italy). All other
organic and inorganic reagents were of analytical grade.
Ferro fluid (FF) synthesis
A solution of FeCl3.6H2O (0.5 M) and FeCl2.4H2O (0.5 M)
mixed in a molar ratio of 2:1 was prepared in contact with
air. An ammonia aqueous solution (25%) of 15 ml was then
quickly charged into the solution using mechanical stirring
against air until the pH value of the solution reached 11. Oleic
acid (5% v/v) was added and intensely stirred at 60◦C for
30 min. The precipitate was separated using a magnet and
washed with deionized water several times. The FF solution
was centrifuged and the appropriate amount of solid phase
was dispersed in kerosene .
Synthesis of chitosan-pectin polyelectrolyte complex
Chitosan hydrochloride salt (10 g, containing 1.00 g chitosan
and stoichiometric amount of HCl) and saturated NH4HCO3
solutions were mixed and incubated at 20◦C for 5 days with
no stirring to obtain chitosan carbamate, Chit-NHCO−2 NH
This chitosan carbamate ammonium was poured into a four-
fold weight of water, and stirred for 30 s with an emulsifier
to obtain a clear solution. Polygalacturonic acid (pectin) was
dissolved in dilute NH4HCO3 solution. The two solutions
were mixed just before spray-drying .
Preparation of magnetic chitosan microspheres
A 200 ml of chitosan-pectin dispersion were added 10 ml of
FF and was stirred vigorously for 2 hours. After that, part
of the dispersion was dehydrated in a spray dryer to obtain
chitosan-pectin encapsulated FF (CPFF) microspheres.
Enzyme immobilization by entrapment: 2 gr of man-
nanse were added to the other fraction of chitosan-pectin-
FF aqueous system. The resulting dispersion was stirred
during 24 h. Next, it was passed through the spray dryer
keeping the dried product temperature below 70◦C to pre-
vent thermal inactivation of the enzyme. The encapsu-
lated Chitosan/Pectin/FF/Mannanase biocatalyst was coded
Covalent enzyme immobilization
The CPFF magnetic microspheres were contacted with
buffered 2.0% glutaraldehyde solution at pH 7during 4 h.
Finally, the glutaraldehyde-treated particles were rinsed and
contacted with a buffered concentrated mannanase enzyme
solution at pH 6 (room temperature, 24h). After natural dry-
ing there were obtained an immobilized mannanase coded
Elemental analysis of supports and immobilized mannanase
systems was conducted using Energy Dispersive X-ray
(EDX) in six micro-zones of each sample by means of an
ESEM/EDX XL30 TMP Philips, 20 KV accelerator voltages.