Immobilization of β-Galactosidases on Magnetic Nanocellulose: Textural, Morphological, Magnetic, and Catalytic Properties

Article abstract of DOI:10.1021/acs.biomac.9b00285

We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-NC) or alkaline treatment (OH-NC) of microcrystalline cellulose, which was subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) β-galactosidases. The mean size of magnetic nanocellulose particles was approximately 75 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with a coercivity of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose either in permeate or in cheese whey for 30× and it reached hydrolysis higher than 50%. Furthermore, using a continuous process in a column packed-bed reactor, the derivative OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or whey after 24 h of reaction. Fungal β-galactosidases immobilized on magnetic nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.

Full text of DOI:10.1021/acs.biomac.9b00285

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Article
Biocatalysts #-galactosidases on magnetic nanocellulose:
textural, morphological, magnetic and catalytic properties
Adriano Gennari, Francielle H. Mobayed, Brenda Da Rolt Nervis, Edilson Valmir Benvenutti,
Sabrina Nicolodi, Nadya Pesce da Silveira, Giandra Volpato, and Claucia F. Volken de Souza
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00285 • Publication Date (Web): 14 May 2019
Downloaded from http://pubs.acs.org on May 21, 2019
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Biocatalysts β-galactosidases on magnetic nanocellulose: textural, morphological,
magnetic and catalytic properties
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Adriano Gennari , Francielle H. Mobayed , Brenda Da Rolt Nervis , Edilson V.
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Benvenutti Sabrina Nicolodi , Nádya Pesce da Silveira , Giandra Volpato , Claucia F.
,
Volken de Souzaa*
a
Laboratório de Biotecnologia de Alimentos, Programa de Pós-Graduação em
Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil.
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Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS,
Brazil.
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Instituto de Física, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS,
Brazil.
d
Curso de Biotecnologia, Instituto Federal de Educação, Ciência e Tecnologia do Rio
Grande do Sul - IFRS, Campus Porto Alegre, Porto Alegre, RS, Brazil.
Abstract
We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-
NC) or alkaline treatments (OH-NC) of microcrystalline cellulose, which was
subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and
used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-
NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) β-
galactosidases. The mean size of magnetic nanocellulose particles was approximately
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5 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with coercivity
of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose
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either in permeate or in cheese whey for 30x and it reached hydrolysis higher than 50%.
Furthermore, using a continuous process in a column packed bed reactor, the derivative
OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or
whey after 24 h of reaction. Fungal β-galactosidases immobilized on magnetic
nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.
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Keywords: Aspergillus oryzae, Kluyveromyces lactis, batch hydrolysis, packed bed
reactor.
1 Introduction
Cellulose is a renewable biomaterial, which has been studied with several
different practical approaches. It is one of the most abundant polymers on Earth, and it
might be obtained from several sources, such as timber, cotton, and other plant-based
materials.1–3 Cellulose is chemically inert and has no toxicity, which enables its
application together with other biomaterials. Its chemical structure has hydrophilic and
hydrophobic characteristics, and this polysaccharide also has a series of hydroxyl
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groups on its surface that might react with several compounds. Cellulose derivatives,
such as carboxymethylcellulose (CMC), cellulose acetate (CA), and microcrystalline
cellulose (MC), are low-cost, biodegradable, and biocompatible substances, and have
therefore raised interest of chemical and biological industries for several applications.5,6
With the recent development of nanotechnology, several types of materials with
nanometric size, such as metallic nanoparticles, carbon nanotubes, nanofibers, and
nanocellulose have been used to enhance the physical and mechanical properties of
biopolymers.7,8 Several techniques have been studied for the preparation of these
materials, which might be via synthesis, e.g. for the production of magnetic
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nanoparticles, or via acidic or alkaline hydrolyses, to obtain nanofibers or
nanocellulose.9–11 Ion oxide nanoparticles are the most explored magnetic nanoparticles
in the industrial and biotechnological fields. Since iron particles can be nanometric, they
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become superparamagnetic, and thus avoid self-agglomeration. These nanoparticles
are inexpensive to produce, have sufficient physical and chemical stability, as well as
biocompatibility, and are environmentally safe. In addition, they show their magnetic
behavior only when an external magnetic field is applied.13 Magnetic iron oxide
particles have been used with nanocellulose in several applications, such as dye
extraction,14–16 drug delivery system, removal of ion pollution, nanocatalyst of 2-
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oxodihydropyrrole derivatives,19 monitoring of emerging pollutants,20 and
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neuro‐endovascular treatment. Aside from iron, other metals have been used for the
development of nanoparticles, e.g. nickel,22 cobalt,23 or manganese,24 as well as
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mixtures of several metals, e.g. zinc, iron, and zircon, which have been applied in the
separation of proteins, therapeutic release of DNA and medications, and in catalytic
processes.
Enzyme immobilization is a biotechnological process employed to reduce some
disadvantages of the use of enzymes in industrial processes, mostly related to their
instability at different ranges of their optimal reaction conditions (pH and temperature)
and to the difficult recovery and reuse of these biocatalysts.26 However, although
several immobilization methods have already been studied, there has still been constant
search for materials that are low cost, easy to obtain, stable, nontoxic, resistant to
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mechanical force, and biocompatible without compromising protein structure. Other
factors about supports that must also be observed are composition, functional groups,
hydrophobicity, morphology, particle size, porosity, active surface area, abrasion (for
stirred tanks), and flow resistance (for a column packed bed reactor).28,29 Since
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nanocellulose has most of these characteristics and its application in the magnetic form
further increases its application potential, we selected this polymer to study β-
galactosidases immobilization.
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β-galactosidase or lactase (β-d-glucopyranosyl-(1→4)-d-glucopyranose) is an
enzyme used primarily in the dairy industry as it catalyzes the conversion of lactose into
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its corresponding sugars, glucose and galactose. The major industrial β-galactosidases
derive from Kluyveromyces lactis, Kluyveromyces fragilis, Aspergillus niger, and
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Aspergillus oryzae. These enzymes are widely applied in the development of products
with reduced lactose contents or lactose-free products, which are intended for
consumption by people incapable of hydrolyzing this sugar.32 β-galactosidases from
different sources have been immobilized on organic and inorganic supports using
several techniques33,34, e.g. Kluyveromyces marxianus and Escherichia coli lactases
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using covalent bond, A. oryzae enzyme using entraptment, K. lactis β-galactosidase by
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crosslinking, and the enzyme derived from Lactobacillus acidophilus by adsorption.
However, there have been no reports to date involving β-galactosidase immobilization
on nanocellulose linked to magnetic particles.
In the present study, we describe a process for obtaining nanocrystalline
cellulose (NC) using either acidic or alkaline treatment, subsequently bonded to
magnetic nanoparticles and used as support for the immobilization of fungal β-
galactosidases of Aspergillus oryzae and Kluyveromyces lactis. Immobilized β-
galactosidases on both magnetic nanocellulose preparations were evaluated regarding
their textural, morphological, magnetic, and thermal stability features, reaction
conditions of pH and temperature, and regarding their kinetic parameters. In addition,
their operational stability was evaluated in lactose hydrolysis in whey and permeate
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using batch processes, and in whey and milk using continuous flow hydrolyses in
packed bed column reactor.
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Material and Methods
.1 Material
Kluyveromyces lactis β-galactosidase (Lactomax Pure) (Free Gal-Kl) and
Aspergillus oryzae β-galactosidase (Lactomax F30) (Free Gal-Ao) were acquired from
Prozyn (São Paulo, Brazil). Ortho-nitrophenyl-β-D-galactopyranoside (ONPG),
Bradford reagent, and microcrystalline cellulose were acquired from Sigma-Aldrich (St.
Louis, USA). The glucose quantification kit was acquired from Labtest (Minas Gerais,
Brazil). Skimmed milk was acquired from Brasil Foods S.A. (Rio Grande do Sul,
Brazil). Cheese whey and whey permeate were provided by Arla Foods (Córdoba,
Argentina). All other reagents used had analytical level.
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.2 Methods
.2.1 Enzyme activity of β-galactosidases
β-galactosidase enzyme activity was determined using the chromogenic
substrate ortho-Nitrophenyl-β-galactoside (ONPG). To analyze enzyme activity, a 50
μL aliquot (soluble or immobilized enzyme) was added to 180 μL ONPG (18 mM) and
270 μL of 100 mM buffer sodium acetate, pH 4.5 (for reactions with the Aspergillus
oryzae enzyme) or 100 mM sodium phosphate with 4 mM of MgCl , pH 7.0 (for
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Kluyveromyces lactis enzyme). The reaction was performed for 2 min at the optimal
temperature of each enzyme (55 °C for A. oryzae or 37 °C for K. lactis). The Ortho-
nitrophenol (ONP) released was determined using spectrophotometer at wavelength of
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15 nm. One unit of β-galactosidase was defined as the amount of enzyme that releases
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μmol of ONP (ε = 4500 L.mol .cm ) per minute under assay conditions. Protein
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content was determined using the method proposed by BRADFORD .
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.2.2 Preparation of nanocellulose-magnetic nanoparticles
To obtain nanocellulose via acidic treatment (H-NC), 10 g of microcrystalline
cellulose were mixed to 250 mL of a 6 M HCl solution and subsequently heated at 90
°C under constant stirring. The reaction was maintained for 90 min, and at the end, the
suspension was immersed in ice bath to interrupt the reaction. Treated cellulose was
separated by centrifugation (2,370 × g or 5,000 rpm, 4 °C, 5 min) and rinsed with
ultrapure water until the mixture reached neutral pH. Finally, the material was
reconstituted at a concentration of 25 mg of cellulose per mL of ultrapure water, and
stored at 4 °C until its further use.
The alkaline treatment of cellulose was performed adding 10 g of
microcrystalline cellulose in 200 mL of 7% NaOH (w/v) and 12% urea (w/v). The
mixture was stirred at 100 rpm for 30 min at 25 °C, and after that, kept at -18 °C for 15
h. After this step, the mixture was homogenized for 10 min in a magnetic agitator, and
the reaction was stopped by adding 1000 mL of ultrapure water. The material obtained
was separated by centrifugation (2,370 × g or 5,000 rpm, 4 °C, 5 min) and rinsed 6 to 7
times with ultrapure water to remove the urea and NaOH and reduce pH to ~7. The
nanocellulose obtained via alkaline medium (OH-NC) was stored in an aqueous solution
at 4 °C at a concentration of 25 mg/mL.
Magnetic nanoparticles (MNP) were synthesized using the chemical co-
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precipitation method . Iron oxides (FeCl .6 H O; 4.2 g and FeCl .4 H O; 2.16 g) were
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dissolved in 200 mL of ultrapure water under an atmosphere of nitrogen and constant
stirring. The mixture was incubated at 85 °C for 5 min, and after that, 40 mL of NH OH
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P.A. were added. The reaction occurred for 12 h under magnetic agitation, and after
that, nanoparticles were collected using centrifugation (2,370 × g or 5,000 rpm, 4 °C, 5
min) and rinsed 4 times with ultrapure water. MNP were dried in a vacuum oven at 50
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For the nanocellulose obtained from the acidic treatment (H-NC) to bond to
magnetic nanoparticles, 16 mL of H-NC (25 mg/mL) were mixed with 100 mg of MNP,
8 mL of 0.1 M HCl, and 25 mg of sodium tripolyphosphate (TPP). The mixture was
maintained in an ultrasound bath (25 kHz) for 4 h at 25 °C. At the end, the H-NC-MNP-
TPP was separated by centrifugation (2,370 × g or 5,000 rpm, 4 °C, 5 min), rinsed, and
stored in ultrapure water until the immobilization process. The procedure of bonding
nanocellulose to magnetic nanoparticles was also performed without the presence of
TPP, generating H-NC-MNP. The nanocellulose derived from the alkaline treatment
(OH-NC) was bonded to magnetic nanoparticles by mixing 16 mL of OH-NC (25
mg/mL) with 100 mg of MNP, 8 mL of 0.1 M HCl, and 25 mg of sodium
tripolyphosphate (TPP). The mixture was maintained in an ultrasound bath (25 kHz) for
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h at 25 °C. At the end, the OH-NC-MNP-TPP was separated by centrifugation (2,370
× g or 5,000 rpm, 4 °C, 5 min), rinsed, and stored in ultrapure water until the
immobilization process. Similarly, bonding nanocellulose (OH-NC) with magnetic
nanoparticles was also performed without the presence of TPP, resulting in OH-NC-
MNP.
2
.2.3 Immobilization of β-galactosidases on magnetic nanocellulose
The immobilization processes were conducted similarly, using H-NC-MNP-TPP,
H-NC-MNP, OH-NC-MNP-TPP, and OH-NC-MNP and both fungal β-galactosidase
enzymes.
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Immobilization with A. oryzae β-galactosidase was performed by incubating 6
mL of enzyme solution (10-200 mg of protein per gram of support) in a buffer sodium
acetate (100 mM pH 4.5) with 100 mg of each preparation. The mixture was maintained
under stirring of 150 rpm at 25 °C for 3 h, and supernatant aliquots were periodically
collected to evaluate yield and efficiency of the immobilization process. At the end, A.
oryzae β-galactosidase immobilized on acidic magnetic nanocellulose (H-NC-MNP-
TPP-Ao and H-NC-MNP-Ao) and on alkaline magnetic nanocellulose (OH-NC-MNP-
TPP-Ao and OH-NC-MNP-Ao) were separated by centrifugation (2,370 × g or 5,000
rpm, 4 °C, 5 min). Derivatives were rinsed with buffer sodium acetate (100 mM pH 4.5)
to remove the non-bonded enzyme, and maintained in the same buffer for subsequent
evaluation.
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The immobilization process with K. lactis β-galactosidase was performed by
incubating 6 mL of enzyme solution (10 to 200 mg of protein/g of support) in buffer
sodium phosphate (100 mM pH 7.0 with 4 mM of MgCl ) with 100 mg of each
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preparation. The mixture was maintained under stirring of 150 rpm at 25 °C for 24 h,
and supernatant aliquots were periodically collected to evaluate yield and efficiency of
the immobilization. Finally, K. lactis β-galactosidase immobilized on acidic magnetic
nanocellulose (H-NC-MNP-TPP-Kl and H-NC-MNP-Kl) and on alkaline magnetic
nanocellulose (OH-NC-MNP-TPP-Kl and OH-NC-MNP-Kl) was separated by
centrifugation (2,370 × g or 5,000 rpm, 4 °C, 5 min). Derivatives were rinsed with
buffer sodium phosphate (100 mM pH with 4 mM of MgCl ) to remove the non-bonded
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enzymes, and maintained in the same buffer for subsequent evaluation.
The immobilization processes were evaluated regarding their efficiency and
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yield, according to Sheldon & Van Pelt .
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.2.4 Textural, morphological, and magnetic characterization of supports and
derivatives
Nitrogen adsorption and desorption isotherms of modified supports (H-NC-
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MNP and OH-NC-MNP) and derivatives (H-NC-MNP-Ao, H-NC-MNP-Kl, OH-NC-
MNP-Ao, and OH-NC-MNP-Kl) were obtained at nitrogen boiling temperature, using
the Tristar II Kr 3020 Micromeritics Equipment. Samples were previously macerated
and dried at 40 °C, under vacuum, for 30 h. Specific areas were estimated using the
Brunauer, Emmett and Teller (BET) method, and size distribution of pores was
4
1
estimated using the Barret, Joyner and Halenda (BJH) method .
Structural morphology of samples (MNP, H-NC, OH-NC, H-NC-MNP-Ao, H-
NC-MNP-Kl, OH-NC-MNP-Ao, and OH-NC-MNP-Kl) was observed using a Scanning
Electronic Microscope with Field Emission (SEM-FEG) (Inspect F50, FEI) with an
acceleration voltage of 20 kV and 150,000 x magnification. Elemental composition of
samples was determined using Energy Dispersive Spectroscopy (EDS) with acceleration
tension of 20 kV.
The magnetic properties of cellulose preparations (H-NC-MNP and OH-NC-
MNP) and of the derivatives produced (H-NC-MNP-Ao, H-NC-MNP-Kl, OH-NC-
MNP-Ao, and OH-NC-MNP-Kl) were studied using a vibrating sample magnetometer
(VSM) (EZ9, MicroSense). Magnetization samples were obtained according to the
applied field (M × H), with field scanning between −20 and +20 kOe, at room
temperature.
The zeta potential of free enzymes and their derivatives was determined using
Zeta Plus Brookhaven Instruments at 25 °C. Enzyme preparations were resuspended at a
ratio of 1:5, in a buffer sodium acetate (100 mM, pH 4.5) for A. oryzae β-galactosidase
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and in a buffer sodium phosphate (100 mM, pH 7.0, with 4 mM MgCl ) for K. lactis
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enzyme.
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.2.5 Catalytic characterization of soluble enzymes and derivatives
Optimal conditions of pH for catalysis of enzymes in both soluble (Free Gal-Kl
and Free Gal-Ao) and immobilized forms (H-NC-MNP-Ao, H-NC-MNP-Kl, OH-NC-
MNP-Ao, and OH-NC-MNP-Kl) were investigated by varying pH from 4.5 to 5.5 with
fixed temperature at 55 °C (for A. oryzae β-galactosidase) and varying pH from 6.5 to
7
.5 with fixed temperature at 37 °C (for K. lactis enzyme). In order to check the
operational temperature of enzymes in their soluble and immobilized forms, pH was
kept at an optimal value for each enzyme according to literature 42,43 (4.5 and 7.0; for A.
oryzae and K. lactis, respectively) and temperature varied from 30 to 65 °C.
Kinetic parameters of enzymes in both soluble (Free Gal-Kl and Free Gal-Ao)
and immobilized forms (H-NC-MNP-Ao, H-NC-MNP-Kl, OH-NC-MNP-Ao, and OH-
NC-MNP-Kl) were determined based on the Michaelis-Menten model, using
Lineweaver Burk's linearization. In this stage, the enzyme activity assay was carried out
with variation in substrate concentration ranging from 3 to 18 mM of ONPG. The
-1
turnover (k ) (min ) of enzymes in the soluble and immobilized forms was determined
cat
−
1
using the ratio between Vmax (mM min ) and total enzyme concentration [ET] in the
reactional medium (mM).
Thermal stability was determined incubating soluble and immobilized A. oryzae
β-galactosidase (Free Gal-Ao, H-NC-MNP-Ao and OH-NC-MNP-Ao) at temperatures
of 65, 68, 70, and 73 °C and K. lactis β-galactosidase (Free Gal-Kl, H-NC-MNP-Kl, and
OH-NC-MNP-Kl) at 55, 58, 60, and 63 °C, and determining residual activity of β-
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galactosidase over time. Thermal inactivation parameters were calculated according to a
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simplified deactivation model described in literature .
Activation energy (Ea) for the inactivation process was calculated using a non-
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linear regression and considering Arrhenius equation (Equation 1):
Ea
―
R.T
퐾 = A.e
(1)
-1
-1
where K (min ) is the constant of kinetic deactivation, A (min ) is the pre-exponential
-1
-1 -1
factor, Ea (J.mol ) is the activation energy, R (8.314 J.mol .K ) is the universal
constant of gases, and T (K) is temperature.
Half-life (t ) of the enzyme is defined as the time required for the enzyme to
1
/2
lose half of its initial activity. Half-life was estimated using Equation 2:
t1/2 = ln 2/K
(2)
-1
where t (min) is the half-life time and K (min ) is the constant of kinetic deactivation.
1
/2
The stabilization factor (SF) for each temperature evaluated was calculated by
dividing the half-life time of each immobilized β-galactosidase by the half-life of its
soluble form (Free Gal-Kl or Free Gal-Ao).
2
.2.6 Batch and continuous reusability of derivatives
Reusability of immobilized enzymes (H-NC-MNP-Ao, H-NC-MNP-Kl, OH-
NC-MNP-Ao, and OH-NC-MNP-Kl) was evaluated using hydrolysis tests in batch
processes with cheese whey and whey permeate, both reconstituted at 5% (w/v) of
lactose. The reactions were conducted with 100 mg of derivatives (containing
approximately 84 U for A. oryzae β-galactosidase derivatives and approximately 76 U
for K. lactis β-galactosidase derivatives) in 10 mL of substrate solutions at 4 °C and 150
rpm for 30 min. At the end of each hydrolysis cycle, the immobilized enzyme was
recovered by centrifugation (1,000 × g or 2,100 rpm, 1 min, 25 ° C), and a new solution
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of substrate was added in order to start a new hydrolysis process. The percentage of
lactose conversion was monitored using the colorimeter enzyme kit of oxidase-
peroxidase (BioLiquid) to check the concentration of glucose in the reactional medium
after each hydrolysis reaction.
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The derivative OH-NC-MNP-Ao was used for lactose hydrolysis reactions in
skimmed milk and cheese whey (both reconstituted at 5% (w/v) of lactose) in
continuous process. The continuous processes for lactose hydrolysis were performed in
a column packed bed reactor (h: 13.2 cm, d: 2 cm), which was filled up with 2 g of
derivative (containing approximately 84 U). Hydrolysis assays were performed at 25 °C
for 48 h with feeding outflow of 2 mL/min. Samples were collected at the reactor output
at different time intervals, and lactose hydrolysis was monitored with glucose
quantification assays using the colorimetric enzyme kit of oxidase-peroxidase
(BioLiquid).
The morphology of derivatives after reusability assays was evaluated using a
Scanning Electronic Microscope with Field Emission (SEM-FEG) (Inspect F50, FEI),
with an acceleration voltage of 20 kV and 150,000 x magnification.
3
3
Results and Discussion
.1 Immobilization of β-galactosidases on magnetic nanocellulose
Microcrystalline nanocellulose (NC) was submitted to two treatments to obtain
nanocellulose, one in an acidic medium (H) and another in alkaline solution (OH)
Figure 1). Subsequently, NC was bonded to magnetic nanoparticles (MNP). The use of
(
tripolyphosphate (TPP) as a reticulating agent was also evaluated in each process.
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Figure 1. Diagram of cellulose hydrolysis processes via the acidic treatment and the
alkaline treatment.
Figure 2 shows the amount of immobilized protein in each treatment according
to each concentration evaluated (10 to 200 mg/g support) of A. oryzae β-galactosidase.
In amounts of up to 100 mg of protein, practically all enzyme was immobilized,
regardless of the type of NC treatment. In the derivatives obtained from cellulose via
acidic medium (Figures 2a and 2b), immobilization occurred more rapidly, reaching
yields similar to their maximum values after approximately 40 min. On the other hand,
derivatives with cellulose obtained from the treatment with NaOH (Figures 2c and 2d)
reached the same values in approximately 120 min. For the evaluated load of 200 mg of
protein, approximately 50% of the enzyme was immobilized. Efficiencies of the
derivatives with enzymes obtained from filamentous fungus, at a load of 100 mg of
protein, were 96.45, 98.34, 93.55, and 94.77% for derivatives H-NC-MNP-Ao, H-NC-
MNP-TPP-Ao, OH-NC-MNP-Ao, and OH-NC-MNP-TPP-Ao, respectively. The results
45
of immobilization parameters were higher than those found by Guerrero et al. , who
studied the immobilization process of Aspergillus oryzae β-galactosidase on glyoxyl-
agarose. These authors obtained with a protein load of 30 mg/g of support, yield and
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efficiency of 50 and 30%, respectively. These results were attributed to the chemical
structure of the support, which will affect its load capacity and the level of its
interaction with the enzyme.
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Figure 2. Quantity of immobilized enzyme with different loads of Aspergillus oryzae β-
galactosidase into (a) H-NC-MNP-Ao, (b) OH-NC-MNP-Ao, (c) H-NC-MNP-TPP-Ao,
and (d) OH-NC-MNP-TPP-Ao. () 10 mg of protein, () 50 mg of protein, () 100
mg of protein, and () 200 mg of protein.
Immobilization curves with different protein loads of K. lactis β-galactosidase
(Figure 3) were evaluated with protein concentrations of 10 to 200 mg/g support.
Similar to what was observed with A. oryzae enzyme (Figure 2), immobilization
occurred more rapidly in derivatives with NC obtained via the acidic treatment.
However, maximum yields were reached only after 24 h of process. The 200 mg
enzyme load was not completely immobilized during this period, with yields around
4
0%. On the other hand, using half of this amount of protein (100 mg), yields for the
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four derivatives ranged between 78 and 83%, while efficiencies were higher than 96%.
Moreover, the use of TPP did not result in differences in amount of immobilized protein
at the end of the period of the study process for either enzyme studied.
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Figure 3. Quantity of immobilized enzyme with different loads of Kluyveromyces lactis
β-galactosidase into (a) H-NC-MNP-Kl, (b) OH-NC-MNP-Kl, (c) H-NC-MNP-TPP-Kl,
and (d) OH-NC-MNP-TPP-Kl. () 10 mg of protein, () 50 mg of protein, () 100
mg of protein, and () 200 mg of protein.
De Albuquerque et al.46 evaluated the immobilization process of this same
enzyme, K. lactis β-galactosidase, on chitosan particles. They showed that by using 10
mg of protein/g support, approximately 100% of the K. lactis β-galactosidase was
immobilized on the support activated by the glutaraldehyde, while less than 40% of
enzyme was immobilized on non-activated chitosan. Regarding the efficiency of the
processes, values of approximately 65% were observed in both. Based on these data, the
authors reported the importance of support treatment, with addition of chemical groups
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to the surface of the support. In the present study, although cellulose was not submitted
to activation processes, protein loads higher than those used by De Albuquerque et al.46
were used, and higher immobilization results were obtained. These differences in
behavior between the supports, chitosan and cellulose, are probably due to size and
surface area of particles of the materials employed.
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A study conducted by Rodriguez et al.47 analyzed the immobilization of β-
galactosidases on magnetic nanoparticles. The protein load employed was lower than 30
mg/g of support. Our study has allowed for the evaluation of loads of up to 200 mg/g,
and both immobilization yield and efficiency were high with half of this load. This
higher immobilization ability was probably enabled because nanocellulose was
48
obtained. According to Klein et al. , the physical characteristics of the support, such as
particle size and pore size, determine its surface area, thus affecting load capacity and
3
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determining the impact of diffusional restrictions. Henriques et al., who also used
magnetic nanoparticles (MNP), evaluated the co-immobilization of lipases and β-
galactosidases. Initially, Thermomyces lanuginosus (TLL) lipase was immobilized on
nanoparticles, which afterwards were used for Kluyveromyces lactis (βGal) β-
galactosidase immobilization. Thereafter, the co-immobilized derivative MNP-TLL-
βGal was crosslinked with glutaraldehyde and dextran-aldehyde. The immobilization
yield of all derivatives was 97%, and recovery activities were between 50 and 93%. The
authors observed that βGal cannot be immobilized directly on the magnetic support
without any surface modifications, and this type of support modification is much more
complicated than using an aminated lipase as an anchor for βGal. In our study, the use
of cellulose with magnetic nanoparticles was a simple but effective method to allow the
bonding of β-galactosidases, which provided recovery activities of about 79 and 94%
for K. lactis and A. oryzae derivatives, respectively.
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The difference in immobilization time observed for the same enzyme in the
different cellulose treatments might probably be related to the final NC structure in each
process. Nanocellulose was obtained by using either acidic or alkaline treatments that
resulted in hydrolyses of the bonds α(1→4), or β-elimination reactions, respectively.
The acidic hydrolysis reaction occurred by depolymerization (reduction in size of the
polymeric chain) due to the temperature employed and the use of acidic medium,
resulting in hydrolysis of α (1→4) bonds of cellulose 49. In β-elimination (alkaline
treatment), the more acidic hydrogen of the cellulose molecule reacts with hydroxide
ion, resulting in the breaking of the glycosidic bond and in the formation of a double
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conjugated bond due to electron transfer. Considering these nanocellulose forms, β-
galactosidase immobilization occured via different physical adsorptions. This process
probably involved hydrogen bonds between hydroxil groups and the hydrogen bond
network in cellulose, respectively, with amine and carboxyle groups of enzyme amino
5
1
acid side chains. The use of magnetic particles might have further contributed with the
electrostatic interaction between β-galactosidases and magnetic nanocellulose.52
Moreover, cellulose hydrophobic groups could also be involved in interactions with
enzymes.53
54
The use of nanosupports of cellulose was also studied by Bansal et al. , who
evaluated the characteristics of laccase enzyme immobilized on cellulose nanofibers
extracted from pomace. Cellulose nanofibers were activated by a single treatment with
polyethyleneimine, glutaraldehyde, and Cu(II). The authors carried out the
immobilization process of laccase with a load of 300 U/g of support and obtained a
3
4
yield of 85%. Chen and Duan , on the other hand, studied β-galactosidase
immobilization on chitosan-coated magnetic Fe O nanoparticles (Fe O -CS).
3
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3
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Immobilized enzyme was prepared either with or without the coupling agent,
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tris(hydroxymethyl)phosphine (THP). Although the protein load used was lower than
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4
that used by Bansal et al. , the two immobilized systems provided a yield of 40 and
68% with Fe O -CS-THP and Fe O -CS, respectively. In the present study, using an
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intermediate protein load, immobilization yields were higher than 90% with A. oryzae
enzyme and were approximately 80% with K. lactis enzyme.
Based on the results obtained in immobilization curves, the derivatives that did
not contain TPP were selected for characterization experiments, since the use of this
reticulating agent did not result in better immobilization parameters, with the protein
load of 100 mg/g of support.
3
.2 Textural and morphological characterization of supports and derivatives
The surface area and volume of pores in magnetic nanocellulose (NC-MNP) and
of derivatives with A. oryzae and K. lactis β-galactosidases were obtained via
adsorption and desorption isotherms, and are shown in Table 1. NC-MNP was observed
to have the same surface area profile regardless of the protocol used for obtaining them.
In addition, after the immobilization of both enzymes, the similarity between these
values was maintained. Furthermore, higher reduction in surface area was observed
when K. lactis β-galactosidase was immobilized. This is likely associated with the size
of these proteins, since A. oryzae enzyme has 90 kDa while K. lactis enzyme has 119
kDa 55,56
.
Table 1. Texture properties of nanocellulose obtained and of derivatives produced.
Surface Area Volume of pores
Sample
2
-1
3 -1
(
± 1 m g )
(± 0.001 cm g )
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H-NC-MNP
H-NC-MNP-Ao
H-NC-MNP-Kl
OH-NC-MNP
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0.048
0.036
0.019
0.049
0.039
0.021
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OH-NC-MNP-Ao
OH-NC-MNP-Kl
Textural analysis is shown in Figure 4 and Table 1. Isotherms are typical of
mesoporous materials, with high relative pressure hysteresis (Figure 4a). Size
distribution of pores shown in Figure 4b confirms the presence of mesopores between 3
and 20 nm diameter. Enzyme immobilization is observed to result in decreased porosity
in both celluloses obtained (OH-NC-MNP and H-NC-MNP). There is an evident
reduction in volume of absorbed gas, both in adsorption isotherms and in size
distribution curves of pores, which concurs with the specific area values and pore
volumes shown in Table 1. It is possible to infer that the enzyme partially occupies
pores or blocks their access. In both cases, it confirms their immobilization on the
surface of both celluloses.
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Figure 4. Textural analysis of nanocellulose before and after immobilization. (a)
Adsorption and desorption nitrogen isotherms and (b) size distribution of pores. () H-
NC-MNP, (►) H-NC-MNP-Ao, () H-NC-MNP-Kl, () OH-NC-MNP, () OH-NC-
MNP-Ao, () OH-NC-MNP-Kl.
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Jain et al.27 reported the immobilization of horseradish peroxidase enzyme
(HRP) on mesoporous activated carbon with modified surface. Evaluating textural
characteristics, the authors observed a reduction in surface area after enzyme
immobilization, as well as in the micropore and mesopore areas by approximately 25,
30, 25%, respectively. According to the authors, the level of reduction in surface area
indicates that HRP molecules do not occupy all the space of the mesopore, and mostly
pores with dimensions within their size range were immobilized.
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Figure 5. Scanning Electronic Microscopy with Field Emission (SEM-FEG) of the
derivatives produced: (a) MNP, (b) H-NC, (c) OH-NC, (d) H-NC-MNP-Ao, (e) H-NC-
MNP-Kl, (f) OH-NC-MNP-Ao, and (g) OH-NC-MNP-Kl.
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Morphological characteristics of MNP, H-NC, OH-NC, and magnetic
nanocellulose after β-galactosidase immobilization were investigated using SEM-FEG,
as shown in Figure 5. It was possible to observe that microcrystalline nanocellulose
treated with both methods (acidic or alkaline treatments) resulted in the reduction of
micrometric size to a nanometric scale. NC-MNP bonded to β-galactosidases have a
mean size of 75 ± 4 nm and different enzymes employed did not result in morphological
differences; particles showed a similar rounded shape in all treatments. It is also
observable that particles formed an aggregated structure, which might be attributed both
to the process of preparing images and to the natural attraction of particles due to
magnetism.
5
7
Habibi , who also aimed at preparing a magnetic material, studied the
characteristics of a magnetic nanocomposite of cellulose fibers. Using a method of
synthesis of magnetic particles similar to the one in the present study, the author
determined a mean particle size of 33 nm in the morphological evaluation. This smaller
dimension observed compared to our study is probably associated to the different
methods employed to obtain nanocellulose: while Habibi's reactions were carried out
with tosyl chloride, triethylamine, triethanolamine, and LiCl, ours were performed
either with NaOH and urea or with HCl.
Table 2 shows the results of element composition of samples before and after
immobilization. Regarding the composition of materials used in the preparation of
supports, iron was the major component in MNP structure, and the composition of
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cellulose preparations (H-NC and OH-NC) is divided between carbon and oxygen. The
presence of carbon and oxygen was observed in all samples, since these are elements
typical of cellulose. In addition, the bond between MNP and nanocellulose was
confirmed by the high concentration of iron in the samples. Overall, it was possible to
observe that the proportion of Fe is reduced after immobilization of both β-
galactosidases, and this behavior is attributed to the incorporation of carbon and oxygen
derived from protein structures. Obtaining nanocellulose using the alkaline treatment
provided a better efficiency in the bond with magnetic particles, and prior to
immobilization, the preparation OH-NC-MNP contained a higher iron mass
concentration than the sum of concentrations of carbon and oxygen.
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Table 2. Element composition of the magnetic nanoparticles, obtained nanocellulose
and produced derivatives.
Sample
MNP
Carbon (%)
11.06
Oxygen (%)
27.98
Iron (%)
60.96
0.00
H-NC
82.12
17.88
OH-NC
78.50
21.50
0.00
H-NC-MNP
H-NC-MNP-Ao
H-NC-MNP-Kl
OH-NC-MNP
OH-NC-MNP-Ao
OH-NC-MNP-Kl
20.24
44.82
34.94
22.31
24.37
50.91
39.68
36.56
35.34
42.35
37.11
38.52
21.97
27.12
31.19
29.13
33.18
30.26
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The effect of β-galactosidase immobilization on the magnetism present in
different nanocellulose preparations was evaluated regarding properties and magnetic
hysteresis (Figure 6). Magnetic nanocellulose, either acidic or alkaline, was observed to
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have higher saturation magnetization values (M ), 18.6 and 14 emu/g, respectively,
s
compared to their derivatives. After the immobilizations, derivatives of K. lactis
enzyme had higher reduction both in saturation magnetization and in coercive field.
Moreover, coercive values in nanocellulose obtained via alkaline treatment remained
practically constant (~4 kOe) after immobilizations. In all derivatives, ferromagnetic
particles might be classified as soft since they have a narrow hysteresis cycle. This
behavior is a result of the nanometric size of cellulose magnetic particles (75 ± 4 nm).58
With these results, β-galactosidase immobilization was proved not to interfere in the
magnetic response of materials. Small reductions observed are attributed to increased
non-magnetic mass in particles, derived from enzyme molecules, compared to the mass
of magnetic particles. However, nanocellulose obtained via alkaline treatment seems to
have a lower effect on the magnetic characteristics of the Fe particles synthesized. This
might possibly be related to the final cellulose structure obtained in each medium, since
the hydrolysis of α(1→4) bonds (acidic treatment) and β-elimination reactions (alkaline
treatment) create structures with different electronic characteristics.
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Figure 6. Magnetic properties of magnetic nanocellulose obtained via acidic or alkaline
treatments and of derivatives of A. oryzae and K. lactis β-galactosidases. () H-NC-
MNP, () OH-NC-MNP, () H-NC-MNP-Ao, () OH-NC-MNP-Ao, () H-NC-
MNP-Kl, () OH-NC-MNP-Kl.
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Cabrera et al. studied the preparation of a compound made of magnetic diatom
earth (mDE) coated with polyaniline (mDE@PANI). The material was used as a matrix
to immobilize the enzymes invertase, β-galactosidase, and trypsin. Regarding magnetic
properties, the saturation magnetization values found were 12.4 and 11.3 emu/g for
mDE and mDE@PANI, respectively. Hence, the authors showed that there was no
substantial change in the magnetic properties of mDE after coating with polyaniline.
Surface effects, such as a magnetic inactive layer due to the presence of non-magnetic
matter, e.g. cellulose in our study, could be responsible for decreased magnetization
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values .
The samples Free Gal-Ao, H-NC-MNP-Ao, and OH-NC-MNP-Ao had 10.90,
.23, and -5.89 mV of zeta potential, respectively. Preparations with K. lactis enzyme,
5
on the other hand, had -10.64, -3.83, and -7.84 mV of potential in Free Gal-Kl, H-NC-
MNP-Kl, and OH-NC-MNP-Kl, respectively. The fact that the buffer solution used in
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A. oryzae enzyme immobilization had pH 4.5 resulted in a potential with more positive
loads, compared to zeta potentials of K. lactis β-galactosidase, immobilized in buffer
solution with pH 7.0, which only had negative values. Since the zeta potential was
determine at an optimal pH value for each enzyme, this change in loads is probably
related with NC preparation form. Enzymes immobilized on OH-NC, obtained through
alkaline reaction, had lower zeta potential values than those immobilized on H-NC,
generated through acidic hydrolysis. These results show that the concentration of
enzyme molecules used in the immobilization process (100 mg protein/g support) was
insufficient to bond with all reactive groups in cellulose, whether they were positively
or negatively charged, which defined the zeta potential loads of derivatives. Similar
results were observed by Souza et al,61 who studied the molecular bond between
alginate and A. oryzae β-galactosidase. The four derivatives (H-NC-MNP-Ao, OH-NC-
MNP-Ao, H-NC-MNP-Kl, and OH-NC-MNP-Kl) had absolute zeta potential values of
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up to 10 mV, which were similar to those observed by Talbert et al. These authors
evaluated A. oryzae β-galactosidase immobilization on polystyrene microspheres with
chitosan and observed that zeta potential increased from -42 mV to 4 mV after bonding
with the enzyme. According to the authors, low potential values (up to ± 10 mV)
indicate a tendency towards microsphere aggregation.
3.3 Catalytic characterization of soluble enzymes and derivatives
Compared to optimal catalysis conditions (Table 3), while the immobilization of
A. oryzae β-galactosidase on nanocellulose obtained via acidic hydrolysis (H-NC-MNP)
did not result in change in optimal pH of the enzyme, its operational temperature rose
by 5 °C. On the other hand, when the same enzyme was immobilized on nanocellulose
obtained using the alkaline treatment (OH-NC-MNP), both optimal pH and temperature
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values changed. K. lactis β-galactosidase was less susceptible to operational changes,
and its optimal pH did not change in either derivatives, and only the immobilized
enzyme OH-NC-MNP had a rise of 3 ºC in optimal temperature. Changes in optimal
conditions might be attributed to the different interactions between enzyme and
cellulose, mostly through hydrogen bonds63,64. Similar results were observed by Cao et
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al. , who immobilized a Pseudomonas cepacia lipase on magnetic nanocrystals of
cellulose and observed a small change in the optimal conditions of pH (6.0 to 6.5) and
temperature (35 to 40 °C) after immobilization.
Table 3. Optimal catalytic conditions of pH and temperature, and kinetic parameters of
β-galactosidases immobilized on cellulose.
Optimal Conditions
Vmax
kcat
(min-1)
8.66
7.38
4.12
1.92
1.58
1.29
Sample
K (mM)
M
-1
pH
Temperature (°C)
(mM.min )
50.47 ± 4.78
39.31 ± 3.82
31.06 ± 2.83
18.48 ± 2.25
Free Gal-Ao
H-NC-MNP-Ao
OH-NC-MNP-Ao
Free Gal-Kl
4.5
4.5
4.8
6.5
6.5
6.5
58
63
60
40
40
43
5.82 ± 0.68
5.33 ± 0.73
7.55 ± 0.68
9.65 ± 1.22
H-NC-MNP-Kl
OH-NC-MNP-Kl
10.97 ± 2.64 17.28 ± 3.82
13.73 ± 1.26 17.69 ± 0.18
The constant of Michaelis (K ) shown in Table 3 indicates that the affinity
M
between enzyme and substrate was hardly affected in derivatives with H-NC-MNP, and
of A. oryzae and K. lactis enzyme derivatives with OH-NC-MNP showed a reduction by
approximately 30 and 42%, respectively. The maximum reaction velocity, in turn, was
reduced in derivatives of A. oryzae enzyme by 22 and 38% for H-NC-MNP-Ao and
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OH-NC-MNP-Ao, respectively. As for K. lactis β-galactosidase, both Vmax and kcat
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values remained similar to the soluble enzyme. Song et al. , who studied a new
immobilization process of K. lactis β-galactosidase, built a micro-reactor with the
enzyme immobilized on nanotubes of carbon using different approaches. The authors
observed that the affinity between enzyme and substrate and the maximum reaction
velocity did not change after immobilization. These results are similar to those of the
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present study, and show that there was no problems in transfer of mass by the system in
these immobilization processes, more likely due to the use of a nanometric support 67.
Table 4. Thermal properties of soluble and immobilized β-galactosidases.
6
5 °C
68 °C
70 °C
Sample
K
t1/2
K
t1/2
K
t1/2
SF
-
SF
-
SF
-
-
1
(min-1) (min)
(min-1) (min)
(
min ) (min)
Free Gal-Ao
H-NC-MNP-Ao
OH-NC-MNP-Ao
0.17
0.13
0.11
4.08
0.38
0.27
0.24
1.82
0.57
0.42
0.37
1.22
5.33 1.31
6.30 1.55
2.57 1.41
2.89 1.58
1.65 1.36
1.87 1.54
55 °C
0.67
58 °C
0.43
60 °C
0.37
Free Gal-Kl
H-NC-MNP-Kl
OH-NC-MNP-Kl
1.03
0.82
0.77
-
1.62
1.36
1.28
-
1.89
1.41
1.35
-
0.85 1.26
0.90 1.34
0.51 1.19
0.54 1.27
0.49 1.34
0.51 1.40
SF: stabilization factor.
Thermal stability of immobilized enzymes was evaluated using the parameters
kinetic constant of thermal inactivation (K) and half-life time (t ) (Table 4). The
1/2
conditions under which these processes were tested were approximately 12 and 20 °C
higher than optimal temperature values of A. oryzae and K. lactis enzymes, respectively.
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The values of t1/2 were observed to reduce with increased temperatures. After the
immobilization process, both A. oryzae and K. lactis β-galactosidases had higher
thermal stability than their soluble forms. Both enzymes showed increase in t1/2 when
immobilized on H-NC-MNP and OH-NC-MNP. However, nanocellulose obtained via
the alkaline treatment provided higher stability, and enzymes immobilized on this
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support had higher stabilization factors. According to Beck-Candanedo et al. ,
nanocellulose has a unique self-assembling property, which results in improved thermal
69
stability. As reported by Maleki and Kamalzare , cellulose-based nanocomposites with
inorganic materials, such as Fe O nanoparticles, might provide a well-defined
3
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mesostructure with metallic oxide frames that can protect any biomolecule such as
proteins/enzymes from the denaturation process.
3
.4 Batch and continuous reusability of derivatives
Reusability of immobilized enzymes in batch processes was evaluated in
solutions of whey permeate and cheese whey (Figure 7). In the hydrolysis process of
permeate lactose (Figure 7a), derivatives of A. oryzae β-galactosidase had higher
stability than those of K. lactis. This probably occurred due to the permeate solution pH,
which is around 5.7; the enzyme derived from the filamentous fungus has an optimal
activity in more acidic pH's, whereas β-galactosidase obtained from yeast has a better
catalytic action in neutral values. However, in the hydrolysis process of cheese whey
lactose (Figure 7b), with pH of approximately 6.2 in general, no higher stabilities were
observed for derivatives of K. lactis β-galactosidase, thus indicating that derivatives H-
NC-MNP-Ao and OH-NC-MNP-Ao had an optimal catalytic ability in pH values that
does not correspond to their optimal operational range.
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Figure 7. Operational stability of A. oryzae and K. lactis β-galactosidases immobilized
on nanocellulose. Lactose hydrolysis using immobilized β-galactosidases was
performed in permeate (a) and whey (b), both prepared at 5% of lactose. () H-NC-
MNP-Ao; () OH-NC-MNP-Ao; () H-NC-MNP-Kl; () OH-NC-MNP-Kl.
Comparing reusability results of derivatives synthesized from nanocellulose
obtained either by acidic hydrolysis (H-NC-MNP) or by alkaline treatment (OH-NC-
MNP), similar operational stabilities were observed throughout the reuse cycles. The
four derivatives had high stability after 30 reuses, hydrolyzing over 50% of either the
permeate lactose or the whey lactose. The values found are higher than those reported
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by Rahman et al. , who employed lipase immobilized on magnetic cellulose
nanocrystals, and observed the loss of approximately 50% of the initial enzyme activity
after eight reuse cycles.
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