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Biochemical Engineering Journal 93 (2015) 137141
Contents lists available at ScienceDirect
Biochemical Engineering Journal
jo ur nal home page: www.elsev ier .com/ locate /be j
hort communication
ctive site titration of immobilized beta-galactosidase for
theetermination of active enzymes
eter Jochemsa,b,c, Tobias Muellerc, Yamini Satyawali a, Ludo
Dielsa,b,innie Dejonghea,, Ulf Hanefeldc
Separation and Conversion Technology, Flemish Institute for
Technological Research (VITO), Boeretang 200, 2400 Mol,
BelgiumDepartment of Bioscience Engineering, University of Antwerp,
Groenenborgerlaan 171, 2020 Antwerp, BelgiumBiocatalysis and
Organic Chemistry, Biotechnology, Gebouw voor Scheikunde,
Technische Universiteit Delft, Julianalaan 136, 2628 BL Delft,
Theetherlands
r t i c l e i n f o
rticle history:eceived 16 May 2014eceived in revised form 7
October 2014ccepted 9 October 2014vailable online 23 October
2014
a b s t r a c t
In the present study, an active site titration method is
demonstrated, to determine the amount of activeenzyme
(-galactosidase), immobilized on a support. Two types of supports
were investigated, viz. aminoacrylic resin and a mixed matrix
membrane. Furthermore, 2,4-dinitrophenyl
2-deoxy-2-fluoro--d-galactopyranoside was used as an inhibitor for
the active site titration of immobilized -galactosidaseobtained
from Kluyveromyces lactis. Using the active site titration,
approximately 8.3 mg of active enzyme
eywords:ctive site titrationnzymeeta-galactosidase
mmobilizationeads
was found on 1 g of dried commercially available SPRIN imibond,
which is an amino acrylic resin withcovalently bound -galactosidase
obtained from K. lactis. However, this method, in its present
form,was not effective on the mixed matrix membranes due to the
irreversible partial adsorption of the leav-ing group
(2,4-dinitrophenolate) by the membrane. This observation implied
that it is important toinvestigate interactions between the support
and the used inhibitor and leaving group.
2014 Elsevier B.V. All rights reserved.
embrane
. Introduction
-Galactosidase (lactase, EC 3.2.1.23) is a hydrolase, which
cat-lyzes the hydrolysis and transgalactosylation reactions of
lactosend other -d-galactopyranosides, such as 2-nitrophenyl
-d-alactopyranoside. Industrially lactose is hydrolyzed to
increasehe digestibility of milk or to improve the functional
propertiesf dairy products. Furthermore, -galactosidase is used in
anotherarge industrial process: the synthesis of
galacto-oligosaccharidesGOS), which makes use of the transferase
activity of the enzyme1]. Like many other enzymes, -galactosidase
can also be immobi-ized to extract technical and economic benefits
and enable its longime reuse in industrial reactors [2,3].
In case of enzyme immobilization, it is important to knowhe
amount of (active) enzymes immobilized on the support. In
ost cases, the amount of immobilized enzymes is determined
byndirect methods, mostly by calculating the difference between
he amount of enzymes added to the support and the amount ofemaining
un-immobilized enzymes [4]. Immobilization yieldsn the other hand
are calculated as a ratio between immobilized
Corresponding author. Tel.: +32 14 336907; fax: +32 14
321186.E-mail address: [email protected] (W. Dejonghe).
ttp://dx.doi.org/10.1016/j.bej.2014.10.007369-703X/ 2014
Elsevier B.V. All rights reserved.
activity and the starting activity of the enzyme solution [5].
EFBSection on Applied Biocatalysis also recommends to report
theamount of active enzyme in a given preparation, because it
allowsto normalize the activity of different enzyme preparations
andmake a fair comparison between them [6]. There have been onlya
few studies where the possibility to visualize the enzyme anddetect
its activity, in situ, has been explored. The use of fluores-cence
labelling of proteins (BSA, -lactoglobulin and -globulin)was used
to monitor protein fractionation by ultrafiltration andto control
membrane fouling by Crespo et al. [7]. Later, Mazzucaet al. [8]
developed a new combined method merged from theclassical in situ
detection of enzyme activity and western blot. Thismethod was used
for enzyme -glucosidase (from olive greens) ina capillary
asymmetric polysulphone membrane reactor to deter-mine
simultaneously the enzyme spatial distribution through themembrane
thickness and along the membrane module. Moreover,this method was
also used to determine enzyme activity after theimmobilization
through the observation in the light microscopyboth at low and high
magnification [8].
Active site titration is a powerful tool, which allows the
direct
measurement of the molar concentration of active enzyme in agiven
preparation. This technique makes use of a suicidal sub-strate,
meaning that the substrate blocks the active site renderingit
inactive. Thus, the enzyme can only react with one
substratedx.doi.org/10.1016/j.bej.2014.10.007http://www.sciencedirect.com/science/journal/1369703Xhttp://www.elsevier.com/locate/bejhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.bej.2014.10.007&domain=pdfmailto:[email protected]/10.1016/j.bej.2014.10.007
-
138 P. Jochems et al. / Biochemical Engineering Journal 93
(2015) 137141
sed fo
miaspii
flisTbTtilt2ai[
io
2
2
ocwTMdaSwbcw
Fig. 1. Methodology u
olecule. This substrate can contain a leaving group, which is
eas-ly detected by for example spectroscopy; or for higher
sensitivities
fluorescent leaving group [9]. Active site titration has been
useduccessfully for several free and immobilized enzymes (e.g.
lipases,enicillin G acylase) [10,11]. To the best of our knowledge,
there
s no active site titration method available for the
quantification ofmmobilized -galactosidase.
In case of -galactosidase, 2,4-dinitrophenyl
2-deoxy-2-uoro--d-galactopyranoside is an irreversible inhibitor.
This
nhibitor is a 2-deoxy-2-fluoroglycoside and this class of
glyco-ides is known for its enzyme inhibitors with good leaving
groups.he 2-deoxy-2-fluoroglycosyl enzyme complex gets
accumulated,ecause the rate of hydrolysis of this complex is
extremely slow.his accumulation is due to the fluorine at C-2, that
destabilizeshe transition state for the (de)glycosylation, while
the good leav-ng group accelerates the glycosylation [12,13].
Moreover, thiseaving group is very easily detected and quantified
by UV spec-roscopy, making it suitable for active site titration.
The inhibitor,4-dinitrophenyl 2-deoxy-2-fluoro--d-galactopyranoside
waslready used in several mechanism studies, but to our knowledget
has not yet been used for studying immobilized
-galactosidase1217].
This study investigates the possibilities of employing
thisnhibitor for active site titration using -galactosidase
immobilizedn two different carriers, beads and mixed matrix
membranes.
. Materials and methods
.1. Enzymes and reagents
-Galactosidase from Kluyveromyces lactis (with an activityf
75,000 mol ortho-nitrophenol released min1 g1) was pur-hased from
SigmaAldrich NV/SA (Bornem, Belgium). The enzymeas stored at 4 C
and was used without further processing.
risHCl buffer (50 mM) pH 7 containing 50 mM NaCl and 50 mMgCl2
was used in the experiments. SPRIN imibond galactosi-
ase (covalently bound -galactosidase from K. lactis on
aminocrylic resin, particle size: 0.200.50 mm) was kindly donated
byPRIN S.p.A. (Trieste, Italy). The SPRIN imibond galactosidase
was
ashed 3 times using 4 mL of the TrisHCl buffer per gram of wet
eads. All the other chemicals used in the study were of
analyti-al grade. The membrane used for -galactosidase
immobilizationas a flat-sheet mixed-matrix membrane containing
polysulphone
r active-site titration.
and zirconium dioxide. The water permeability and
molecularweight cut-off of this membrane was 296 L/h m2 bar and
13.8 kDa,respectively. Enzyme immobilization on the membrane was
con-ducted on 1 cm2 membrane piece by submerging it in 0.6 mLenzyme
solution. Details of the immobilization have been
reportedpreviously [3]. The inhibitor 2,4-dinitrophenyl
2-deoxy-2-fluoro--d-galactopyranoside, used in this study, was
synthesized asdescribed before and spectra were in accordance with
the literature[13,18,19].
2.2. Active site titration
The methodology used for active site titration, in this study,is
depicted in Fig. 1. The inhibitor 2,4-dinitrophenyl
2-deoxy-2-fluoro--d-galactopyranoside (15 mg) was dissolved in 15
mLof Tris buffer. Three sets of Eppendorf scale tests were
prepared(each in quadruplicate). First set of Eppendorf tubes were
filledwith 100 L of diluted enzyme solution, second set with a
definedamount of imibond beads and the third set with 1 cm2 piece
ofmembrane. From each of the 3 sets, three tubes (out of each
quadru-plicate) were incubated with 600 L (or 500 L in case of the
freeenzyme) of the inhibitor solution at 20 C for 6 h. After these
6 h,260 L of methanol was added to the samples and they were
incu-bated for another hour at 20 C. The enzymes of the three
remainingtubes of each quadruplicate were directly inactivated by
adding260 L of methanol. After 1 h at 20 C, 600 L (or 500 L in case
ofthe free enzyme) of the inhibitor solution was added to these
sam-ples, which were used as blank. These blanks were incubated
foranother 6 h at 20 C. All incubations were performed on a rotator
toobtain a well-mixed system. After incubation, 120 L 2 M Na2CO3was
added to each Eppendorf tube to enhance the colour devel-opment and
all tubes were vortexed and centrifuged (15,000 g).From each of
these tubes, 3 times 200 L supernatants of each sam-ple or blank
was transferred to a multiwell-plate. The absorbanceof each well
was measured at 9 different places (to reduce varia-tions) using a
microplate reader (Infinite M200 PRO, Tecan GroupLtd., Switzerland)
at 400 nm.
2.3. Protein determination
Protein determination was performed using the Bradfordmethod as
previously described [3].
-
P. Jochems et al. / Biochemical Engineering Journal 93 (2015)
137141 139
F omycg due to
2
ett6be
3
gkinmaaa2faotamwfb
the mixed matrix membranes can be easily scaled up by
adapta-tion of the membrane surface [3]. One issue encountered
duringthe active site titration in case of both beads and membranes
waspartial adsorption of the leaving group 2,4-dinitrophenolate
by
y = 0,0077x + 0,5164R = 0,9759
0
2
4
6
8
10
12
14
nmol
2,4
-din
itrop
heno
l rel
ease
d
ig. 2. Schematic mechanism of the active site titration of
-galactosidase of Kluyveralactosyl complex; 3: the stable
enzyme2-deoxy-2-fluoro galactosyl complex
.4. Dry weight of beads
Before each test, the Eppendorf tubes were weighed and afterach
test the Eppendorf tubes containing the samples were cen-rifuged
(15,000 g) and supernatants were removed. Afterwardshe beads were
dried by placing the open tubes in an oven at0 C for 72 h. Finally
the tubes were weighed again. The differenceetween both
measurements is the amount of dry immobilizednzyme in each
tube.
. Results and discussion
The inhibitor 2,4-dinitrophenyl
2-deoxy-2-fluoro--d-alactopyranoside was used previously, as a part
of theinetic study for the active site titration of
non-immobilized-galactosidase of Escherichia coli [17]. Also
analogues of this
nhibitor were used for performing active site titration on
otheron-immobilized -glycosidases, e.g. -glucosidase [12].
Theechanism of active site titration method is shown in Fig. 2.
The
ctive site of -galactosidase of K. lactis contains two
glutamiccid residues (Glu482 and Glu551) that act as proton donornd
nucleophile site [20]. As the enzyme acts on the
inhibitor,,4-dinitrophenyl 2-deoxy-2-fluoro--d-galactopyranoside,
itorms a stable enzyme2-deoxy-2-fluoro galactosyl complexnd
liberates 2,4-dinitrophenolate [12,13]. The concentrationf the
liberated 2,4-dinitrophenolate can be measured
spec-rophotometrically at 400 nm and corresponds with the amount
ofctive enzymes. The amount of active enzyme was calculated by
ultiplying the liberated molar amount of 2,4-dinitrophenolateith
the molecular weight of the enzyme (119 kDa). As revealed
rom Fig. 3, in case of free enzyme, there is a good
correspondenceetween the protein concentration measured using the
Bradford
es lactis. 1: the empty active site; 2: the formation of the
enzyme2-deoxy-2-fluoro the fluorine at C-2 and the liberated 2
,4-dinitrophenolate.
method and the enzyme concentration found using active
sitetitration. These results confirm that active site titration is
indeed areliable method with the free enzyme.
Similar experiments were performed on the two
differentimmobilized enzyme preparations, SPRIN beads and mixed
matrixmembranes. The SPRIN beads are commercially available
aminoacrylic resin beads and were chosen due to their easy
commercialavailability. The mixed matrix membranes were made in
house andwere chosen because they can be easily loaded with enzymes
andoperated with almost no mass transfer limitations.
Furthermore,
0 500 1000 1500 2000g of BSA equ ivalent s
Fig. 3. The relation between the amount of free enzyme (in BSA
equivalents) addedand the released 2 ,4-dinitrophenolate.
-
140 P. Jochems et al. / Biochemical Enginee
y = 0,0691x + 0,3638R = 0,945
0
2
4
6
8
10
12
14
16
18
0 50 100 150 200 250
nmol
2,4-
dini
trop
heno
lrel
eas e
d
mg of dry beads
Fd
td(bSbaemfbpamHbi4Hg
btpelooRAbwC
TA
a
Hc
1
ig. 4. The relation between the amount of SPRIN imibond
galactosidase (in mg ofry resin) added and the released 2
,4-dinitrophenolate.
he supports. The leaving group 2,4-dinitrophenolate is
releaseduring the hydrolysis of the inhibitor. In case of the SPRIN
beadsFig. 4), this problem of leaving group adsorption could be
solvedy desorbing the 2,4-dinitrophenol compound using methanol.uch
adsorption of the leaving group (p-nitrophenolate) has alsoeen
observed previously by Rotticci et al., where toluene was useds a
desorbing agent [10]. Eventually 8.3 mg/g (dry support)
activenzymes were found on the beads (Table 1). However, in case of
theixed matrix membrane using methanol as a desorbent was inef-
ective. A plausible explanation for this observation could be
theulkiness of the membrane and the limited amount of enzymesresent
on the membranes (Table 1), which resulted in very lowmounts (
-
ginee
[
[
[
[
[
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[
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[
[
P. Jochems et al. / Biochemical En
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site titration of immobilized beta-galactosidase for the
determination of active enzymes1 Introduction2 Materials and
methods2.1 Enzymes and reagents2.2 Active site titration2.3 Protein
determination2.4 Dry weight of beads3 Results and discussion4
ConclusionsAcknowledgementsReferences
