Process
Biochemistry
47
(2012)
1095–1101
Contents
lists
available
at
SciVerse
ScienceDirect
Process
Biochemistry
jo
u
rn
al
hom
epage:
www.elsevier.com/locate/procbio
Immobilization
of
commercial
laccase
on
spent
grain
Andreia
Machado
da
Silva
a
,
Ana
P.M.
Tavares
a
,
Cristina
M.R.
Rocha
b
,
Raquel
O.
Cristóvão
a
,
José
A.
Teixeira
c
,
Eugénia
A.
Macedo
a,∗
a
LSRE
-
Laboratory
of
Separation
and
Reaction
Engineering
-
Associate
Laboratory
LSRE/LCM,
Faculdade
de
Engenharia,
Universidade
do
Porto,
Rua
do
Dr.
Roberto
Frias,
4200-465,
Porto,
Portugal
b
REQUIMTE,
Departamento
de
Engenharia
Química,
Faculdade
de
Engenharia,
Universidade
do
Porto,
Rua
do
Dr.
Roberto
Frias,
4200-465,
Porto,
Portugal
c
IBB-Institute
for
Biotechnology
and
Bioengineering,
Centre
of
Biological
Engineering,
Universidade
do
Minho,
Campus
de
Gualtar,
4710-057,
Braga,
Portugal
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
26
September
2011
Received
in
revised
form
27
March
2012
Accepted
29
March
2012
Available
online
6
April
2012
Keywords:
Brewing
Commercial
laccase
Immobilized
enzyme
Kinetic
parameter
Optimization
Spent
grain
a
b
s
t
r
a
c
t
The
aim
of
this
work
was
to
assess
the
possibility
of
using
beer
spent
grain
(a
byproduct
of
beer’s
brewing
industry)
as
a
carrier
for
laccase
immobilization.
Both
adsorption
(on
spent
grain
–
SG
and
on
digested
spent
grain
– DSG)
and
covalent
binding
(using
glycidol
and
glycidol
followed
by
ethylenediamine
on
DSG)
were
used.
The
effect
of
different
immobilization
conditions
on
the
immobilization
yields
and
recovered
activities
such
as
contact
time,
enzyme
concentration
and
pH
was
evaluated.
For
the
best
conditions,
immobilization
yields,
recovered
activities
and
thermal,
operational
and
storage
stabilities
were
also
evaluated.
Finally,
the
Michaelis–Menten
mechanism
was
applied
and
the
parameter
with
respect
to
ABTS
oxidation
was
determined.
Enzyme
immobilization
on
DSG
led
to
the
best
enzyme
activities
(recovered
activities
as
high
as
90%)
and
to
high
storage
and
operational
stabilities
(10
cycles).
Thermal
stability
was
also
improved
and
the
half-life
of
immobilized
laccase
in
SG
increased
from
0.64
h
to
1.1
h
at
70
◦
C.
©
2012
Elsevier
Ltd.
All
rights
reserved.
1.
Introduction
Over
the
last
decades,
progress
in
understanding
the
environ-
mental
impacts
of
pollutants
has
promoted
the
development
of
new
treatment
technologies.
To
overcome
limitations
of
tradi-
tional
treatment
processes,
research
has
focused
on
the
use
of
enzymes
for
the
elimination
of
a
wide
range
of
contaminants.
In
order
to
improve
the
efficiency
and
cost
of
the
enzymatic
process,
the
enzyme
can
be
reused
by
an
immobilization
technique.
This
capacity
to
retain
the
enzyme
on
a
support
also
allows
an
easy
bio-
catalyst
separation
from
product
permitting
continuous
processes
and
prevents
the
deactivation
of
protein
or
loss
of
activity.
Laccases
(EC
1.10.3.2)
are
multi
copper
oxidases
that
can
catalyse
the
oxidation
of
phenolic
substrates
with
concomitant
reduction
of
O
2
and
do
not
require
hydrogen
peroxide
as
co-
substrate
or
any
cofactor
for
the
enzymatic
reaction
[1].
Due
to
their
capability
of
catalysing
the
oxidation
of
aromatic
compounds,
laccases
are
attracting
increasing
attention
as
potential
industrial
enzymes
in
various
applications,
such
as
pulp
delignification
[2],
dye
decolorization
[3,4],
and
contaminated
water
or
soil
remedia-
tion
[5].
∗
Corresponding
author.
Tel.:
+351
22
508
1653;
fax:
+351
22
508
1674.
E-mail
address:
eamacedo@fe.up.pt
(E.A.
Macedo).
The
choice
of
the
most
suitable
method
to
immobilize
the
desired
enzyme
depends
on
both
the
reaction
and
environmen-
tal
conditions,
the
configuration
of
the
reaction
apparatus,
as
well
as
the
carrier
to
be
used.
Also,
the
choice
of
the
most
suitable
car-
rier
on
which
to
perform
immobilization,
besides
depending
on
the
immobilization
method,
is
influenced
by
its
chemical
properties
(composition;
hydrophilic
or
hydrophobic
nature;
stability
in
the
conditions
of
the
reaction
medium;
etc.),
its
mechanical
stability
and
geometric
properties
(size,
shape,
thickness,
porosity,
etc.),
the
characteristics
of
the
enzyme
to
be
immobilized
(chemical
com-
position,
physical
properties,
structure,
etc.)
and
its
ecological
and
biological
properties
(non-toxic
neither
to
the
environment
or
to
humans;
easily
disposable;
etc.)
[6].
In
order
to
achieve
an
econom-
ically
viable
application
at
industrial
level,
some
works
on
effective
cheap
supports
for
enzyme
immobilization
have
been
reported,
like
functionalized
rice
husk
for
invertase
immobilization
[7]
and
rice
straw
for
lipase
immobilization
[8]
and
coconut
fibre
for
laccase
immobilization
[9].
Spent
grain
(SG)
is
the
most
important
by-product
of
the
brew-
ing
industry
which
makes
it
economically
attractive.
In
Portugal,
SG
is
a
disposable
material
of
the
Unicer
group
(beer
producer)
with
no
associated
costs.
It
is
an
abundant
waste
consisting
of
the
solid
residue
generated
after
mashing
and
lautering
processes
[10].
Its
composition
contains
many
functional
groups
such
as
car-
boxyl,
hydroxyl
and
amino
[11]
that
makes
it
a
potential
support
for
enzyme
immobilization.
Although
large
amounts
are
generated
1359-5113/$
–
see
front
matter
©
2012
Elsevier
Ltd.
All
rights
reserved.
http://dx.doi.org/10.1016/j.procbio.2012.03.021