Revista Mexicana de Fısica 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,∗e-mail: [email protected]
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) for2 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 confirmedspherical morphology. Energy-Dispersive X-Ray Spectroscopy (EDX) was used for the detection of iron in the samples. Additional themagnetization profile were obtained.Mannanase was immobilized on magnetic chitosan microspheres by entrapment and cross-linking with glutaraldehyde. The immobilizationconditions were investigated achieving the conservation of catalytic activity. The catalytic behavior of the immobilized mannanase wasacceptable as compared with the same characteristics of the free enzyme. The magnetic characteristic of these materials allows easy removalof enzyme after use.
Keywords: Magnetic chitosan microspheres; immobilization; entrapment; cross linking.
El quitosano ha sido investigado ampliamente para la inmovilizacion de enzimas. En este estudio, se sintetizaron microesferas magneticas dequitosano (MCM) usando un ferrofluido; para insolubilizar el quitosano se utilizo bicarbonato de amonio; estas microesferas fueron usadaspara la inmovilizacion de enzimas. Una solucion de acido acetico (1%w/v) fue usada como solvente para la dispersion de quitosano (2%w/v),esta dispersion se dejo reaccionar con un ferrofluido (2%v/v) por 2 horas con agitacion continua, se agrego la enzima y despues de un tiempotoda la dispersion fue secada en un secador por aspersion obteniendo microesferas magneticas de quitosano con enzima inmovilizada, la car-acterizacion de las microesferas fue realizada con micrografıas SEM/ESEM confirmando su morfologıa esferica. Espectroscopia de energıadispersa de rayos X (EDX) fue usada para la deteccion del hierro en las muestras. Adicionalmente se obtuvo el perfil de magnetizacion.Una mananasa fue inmovilizada en las microesferas magneticas de quitosano por atrapamiento y entrecruzamiento con glutaraldehido. Lascondiciones de la inmovilizacion fueron investigadas logrando la conservacion de la actividad catalıtica. El comportamiento catalıtico dela mananasa inmovilizada fue aceptable en comparacion con la enzima libre en iguales condiciones. Las caracterısticas magneticas de estematerial permiten remover facilmente la enzima despues de uso para utilizarla nuevamente.
Descriptores: Microesferas magneticas de quitosano; inmovilizacion; atrapamiento; entrecruzamiento.
PACS: 75.75.-c; 75.75.Cd; 75.60.Ej; 75.70.Cn; 75.47.Lx
1. Introduction
One of the major sources of renewable organic matter is thehemicellulose, a complex group of polymers. In the cell wallof plants the mannan is one of the major constituent and theenzymes that degrade it have found applications in the phar-maceutical, food, pulp and paper industries [1].
In the food industry, mannan degrading enzymes maybe used for the maceration of fruit and vegetable materialsand clarification of juices and wines [1], in the extraction ofvegetable oils from leguminous seeds, the viscosity reduc-tion in extracts during the manufacture of instant coffee [2],improvement in the consistency of beer, and biopulping ofwood, 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 andpigs), 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-dry [6].
Many enzymes used in the food industry are quite ex-pensive, but when they are insolubilized by coupling them toan adequate matrix, the resulting biocatalyst may be reusedseveral times, thus lowering the costs [7], immobilizationalso allows better reactions control and permits the designof bioreactors that can be easily incorporated into a continu-ous processing line [8]. The properties of immobilized en-zymes are governed by the properties of both the enzymeand the support material [7,9]. One important attribute forusing a support in the food industry is to be inert and non-toxic. One of the options was chitin and chitosan which offera 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 forspray-drying [11].
In recent years, magnetic carrier technology has showedsignificant attractive for the preparation of immobilized en-zymes. Chitosan can be used as a base material for magneticcarriers [12]. 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 amagnetic field.
The aim of this work was to prepare solublepolyelectrolyte-magnetic nanoparticles complexes in solu-tions suitable for spray-drying to obtain, after drying, waterinsoluble polyelectrolyte-magnetic micro-spheres (PMM).Using two different protocols, PMM were employed to man-ufacture immobilized mannanase that were characterized bymeasurements of their catalytic and magnetic properties.
2. Materials and methods
Reagents and materialsFerric chloride (FeCl3.6H2O), ferrous chloride(FeCl2.4H2O), ammonia and kerosene were all chemicalgrade and chitosan flakes (high molecular weight 602 kDa,degree of deacetylation 76.5%) were obtained from SigmaChemical Co. (St. Louis, MO, United States), Ro-halase GMP (an enzyme preparation that contains man-nanase as main activity ) with a nominal specific activityof 1.000.000 MNU g−1 solid and containing 44% proteinbased on the Bradford protein assay were obtained from ABEnzymes (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 otherorganic and inorganic reagents were of analytical grade.Ferro fluid (FF) synthesisA 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 withair. An ammonia aqueous solution (25%) of 15 ml was thenquickly charged into the solution using mechanical stirringagainst air until the pH value of the solution reached 11. Oleicacid (5% v/v) was added and intensely stirred at 60◦C for30 min. The precipitate was separated using a magnet andwashed with deionized water several times. The FF solutionwas centrifuged and the appropriate amount of solid phasewas dispersed in kerosene [13].Synthesis of chitosan-pectin polyelectrolyte complexChitosan hydrochloride salt (10 g, containing 1.00 g chitosanand stoichiometric amount of HCl) and saturated NH4HCO3solutions were mixed and incubated at 20◦C for 5 days withno stirring to obtain chitosan carbamate, Chit-NHCO−
2 NH+.4
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) wasdissolved in dilute NH4HCO3 solution. The two solutionswere mixed just before spray-drying [14].Preparation of magnetic chitosan microspheresA 200 ml of chitosan-pectin dispersion were added 10 ml ofFF and was stirred vigorously for 2 hours. After that, partof the dispersion was dehydrated in a spray dryer to obtainchitosan-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 stirredduring 24 h. Next, it was passed through the spray dryerkeeping the dried product temperature below 70◦C to pre-vent thermal inactivation of the enzyme. The encapsu-lated Chitosan/Pectin/FF/Mannanase biocatalyst was codedas CPFFMCovalent enzyme immobilizationThe CPFF magnetic microspheres were contacted withbuffered 2.0% glutaraldehyde solution at pH 7during 4 h.Finally, the glutaraldehyde-treated particles were rinsed andcontacted with a buffered concentrated mannanase enzymesolution at pH 6 (room temperature, 24h). After natural dry-ing there were obtained an immobilized mannanase codedCPFFMG.Elemental analysisElemental analysis of supports and immobilized mannanasesystems was conducted using Energy Dispersive X-ray(EDX) in six micro-zones of each sample by means of anESEM/EDX XL30 TMP Philips, 20 KV accelerator voltages.β-Mannanase catalytic activity assaySoluble guar gum (1%; 0.5 mL) was incubated with the im-mobilized enzyme samples (30 mg) at pH 6 and 50◦C for30 min. The reaction was stopped by a thermal shock in anice bath, and then the mixture was centrifuged at 3000 rpm(1500 g) for 10 min and 4◦C. The concentration of reduc-ing sugar in the supernatant was determined using the 3,5-dinitrosalicylic acid method. One unit was defined as theamount of enzyme that could produce 1µmol of reducingsugar (mannose base) for 1 min [15].Magnetic characterizationMagnetic properties of the CPFF, CPFFM and CPFFMGparticles were evaluated using a Versalab-Vibrating SampleMagnetometer Quantum Design Inc.
3. Results
Characterization of microparticlesThe morphology of the magnetic chitosan/pectin micropar-ticles was investigated using SEM and ESEM (Fig. 1). Atthe scanning electron microscope, the microspheres of chi-tosan/pectin/FF (CPFF) and chitosan/pectin/FF/mannanase(CPFFM) were mostly in the diameter range 1-5µm, sim-ilar to other magnetic chitosan microspheres prepared by adifferent method [12]. The particle diameter is important fac-tor for support material. Smaller particles have larger surface-to-volume ratios and larger capacity to bind more enzymes
Rev. Mex. Fis. S58 (2) (2012) 39–43
IMMOBILIZATION OF MANNANASE ON MAGNETIC CHITOSAN MICROSPHERES 41
FIGURE 1. SEM and ESEM micrographs (1000x) of chi-tosan/pectin/FF particles obtained by spray drying. (a) CPFF,(b)CPFFFM and (c) CPFFFMG systems.
on their surface, and substrate and product would give lessrestriction for diffusion [16].
Visible changes in the morphological aspect of the spray-dried chitosan/pectin microspheres due to the mannanase en-trapment (Fig. 1(b)) were observed compared to the unim-
mobilized microspheres (Fig. 1(a)). The sulcal pattern al-ready shown on the surface of the latter almost disappear onthe former immobilized mannanase on CPFF microspheres.Smooth surface microspheres is the most common particlecharacteristic reported for spray dried chitosan and chitosanpolyelectrolyte complexes [12,17,18].
Conversely, the glutaraldehyde treated microspheres losttheir shape in the mannanase immobilizing procedure. InFig. 1(c) the ESEM micrograph of CPFFMG magnetic par-ticles were transformed in a porous plate with some isolatedmicrospheres, probably due to the partial dissolution and ag-gregation of the polyelectrolite envelop of the FF particles, asa consequence of the crosslinking associated to the supportglutaraldehyde activation required for the covalent immobi-lization process.EDX measurementsFor the immobilized CPFF support, CPFFM and CPFFMGimmobilized mannanases the presence of three elements wasconfirmed from X-ray EDX signals (Fig. 2). Carbon andoxygen were derived mainly from the biopolymer entrapmentmatrix and iron is attributed to exposed ferrofluid nanopar-ticles. The cuantitative data of six sets of similar sam-ples demonstrated that the composition of michospheres con-sisted on Carbon, Oxigen and Iron elements mainly. The ironis detected when it was used an accelerating beam voltage of
FIGURE 2. Energy-Dispersive X-Ray Spectroscopy (EDX). Theupper micrography illustrate a sample target area. Bottom: TypicalEDX response showing the relative concentration of C, O and Feelements in the microspheres.
Rev. Mex. Fis. S58 (2) (2012) 39–43
42 L.V. ZULUAGA, O.H GIRALDO, AND C.E. ORREGO
TABLE I. EDX (20 kV) relative content (%) of C,O and Fe elementsin the magnetic support (CPFF) and the immobilized mannanases.
SAMPLE C(%) O(%) Fe(%)
CPFF 67.1 31.5 0.9
CPFFM 68.9 29.7 1.0
CPFFMG 72.0 26.1 1.7
Average content of six micro-zones of each sample
FIGURE 3. Demagnetization curves of the samples coated withchitosan/Pectin.
20 KeV (Table I). As the beam voltage was reduced(2.5 keV), the electrons excite X-rays to at lesser particledepths, the iron signal was not detected (data not shown).These results confirm the success of the chitosan/pectin coat-ing on the iron magnetic oxide particles.
The iron content in CPFFMG is higher than in micro-spheres. A possible explanation is that glutaraldehyde treat-ment reduce (or uncover) the biopolymer coat of the iron par-ticles.
3.1. Magnetization profiles
Figure 3 shows the magnetization profile of CPFF, CPFFMmicrospheres and CPFFMG particles. Along with the in-crease of chitosan/pectin in the samples, their saturated mag-netization decreased accordingly. The saturated magnetiza-tion rates of CPFF and CPFFM were 10 emu/g, and 5 emu/g,respectively. The saturated magnetization of higher iron con-tent was equal to the CPFF (10 emu/g) probably owing to thepartial exposition of ferrofluid particles in this sample. Thesesaturated magnetization values were similar to the reportedones for chitosan microspheres (10 to 20 emu/g) in recentstudies [19,20]. The absence of hysteresis indicates these mi-crospheres are superparamagnetic [21].
3.2. Enzyme activity
The variation of product concentration with time is shown inFig. 4. The results show that the enzyme immobilized by
TABLE II. Kinetic parameters of free and immobilized mannanase
Sample Vmax Km
Free enzyme 0.059 5. 101
CPFFM immobilized mannanase 0.074 5.167
CPFFMG Immobilized mannanase 0.114 10.392
FIGURE 4. Progress curve for the enzyme reaction. (♦) Free en-zyme, () CPFFM immobilized mannanase, (x) CPFFMG immobi-lized mannanase () Blank assay.
entrapment retains 91% of the free enzyme activity, whereasthe enzyme immobilized by covalent immobilization retains81%.
The kinetic constants of Km and Vmax are show inTable II. Immobilization increased inVmax value from0.059 mg/(l min) to 0.074 for CPFFM, and to 0.114 forCPFFMG immobilized mannanase. The Km values of immo-bilized mannanases are higher than that of free mannanase,which means the immobilized enzyme had lower affinity to-wards the sustrate. The increase in Km might be caused bythe steric hindrance of the active site by the support, the lossof enzyme flexibility necessary for substrate binding, or dif-fusion limitation of substrate and products because of non-porous nature of the support [12].
4. Conclusions
In this work it was reported a methodology for the synthesisof coated and chitosan/pectin iron oxide magnetic particles(CPFF) and immobilized mannanase on chitosan/pectin ironoxide magnetic particles (CPFFM) using a spray-drying tech-nique. The CPFF microspheres obtained, with a diameter be-tween 1 to 5µm, were also used for covalent immobilizationof the enzyme using a conventional glutaraldehyde activationprotocol (CPFFMG).
All the particle systems obtained were characterized bySEM/ESEM, EDX techniques. According to these data,the CPFF microspheres were transformed in CPFFMG semi
Rev. Mex. Fis. S58 (2) (2012) 39–43
IMMOBILIZATION OF MANNANASE ON MAGNETIC CHITOSAN MICROSPHERES 43
amorphous magnetic particles probably due to the partial dis-solution of the chitosan/pectin coat by the immobilizationtreatment. This fact was also the main cause of the detectionof a major quantity of iron in this system in EDX measure-ments.
Although in the magnetization assays the CPFF andCPFFM did not reach complete saturation, the saturated mag-
netization values of the samples were considerably lower thanthe reported ones for chitosan microspheres.
Finally, the catalytic behavior of the immobilized man-nanases was acceptable as compared with the same charac-teristics of the free enzyme.
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