Site-selective immobilisation of functional enzymes on to polystyrene nanoparticles

Article abstract of DOI:10.1039/b916773k

The immobilisation of proteins on to nanoparticles has a number of applications ranging from biocatalysis through to cellular delivery of biopharmaceuticals. Here we describe a phosphopantetheinyl transferase (Sfp)-catalysed method for immobilising proteins bearing a small 12-mer "ybbR" tag on to nanoparticles functionalised with coenzyme A. The Sfp-catalysed immobilisation of proteins on to nanoparticles is a highly efficient, single step reaction that proceeds under mild conditions and results in a homogeneous population of proteins that are covalently and site-specifically attached to the surface of the nanoparticles. Several enzymes of interest for biocatalysis, including an arylmalonate decarboxylase (AMDase) and a glutamate racemase (GluR), were immobilised on to nanoparticles using this approach. These enzymes retained their activity and showed high operational stability upon immobilisation. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b916773k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Site-selective immobilisation of functional enzymes on to polystyrene
nanoparticles†‡
Lu Shin Wong,* Krzysztof Okrasa and Jason Micklefield*§
Received 13th August 2009, Accepted 6th November 2009
First published as an Advance Article on the web 23rd December 2009
DOI: 10.1039/b916773k
The immobilisation of proteins on to nanoparticles has a number of applications ranging from
biocatalysis through to cellular delivery of biopharmaceuticals. Here we describe a
phosphopantetheinyl transferase (Sfp)-catalysed method for immobilising proteins bearing a small
12-mer “ybbR” tag on to nanoparticles functionalised with coenzyme A. The Sfp-catalysed
immobilisation of proteins on to nanoparticles is a highly efficient, single step reaction that proceeds
under mild conditions and results in a homogeneous population of proteins that are covalently and
site-specifically attached to the surface of the nanoparticles. Several enzymes of interest for biocatalysis,
including an arylmalonate decarboxylase (AMDase) and a glutamate racemase (GluR), were
immobilised on to nanoparticles using this approach. These enzymes retained their activity and showed
high operational stability upon immobilisation.
orientations and potentially reduced activity. As an alternative,
Introduction
we have recently developed a method for the one-step direct site-
Polystyrene nanoparticles (NPs) have been used in the life sciences
specific and covalent immobilisation of proteins on to a variety of
supports.15 This method uses a phosphopantetheinyl transferase
enzyme Sfp to catalyse the immobilisation of recombinant proteins
bearing the small (11 amino acid residues) “ybbR” tag16 on to
supports derivatised with coenzyme A (CoA). The Sfp catalysed
immobilisation results in the attachment of protein of interest
via the Ser residue of the ybbR-tag to the phosphopantetheine
(Ppant) moiety of CoA (attached to the bulk material via the
S-atom), with the concomitant loss of 3¢,5¢-adenine diphosphate
(Fig. 1). The enzymes immobilised this way retained high levels of
activity.15
for several decades, and antibody-conjugated polystyrene latex is
widely used in agglutination tests for the detection of a variety of
biological analytes.1 However in recent years, improved methods
of preparing NPs with well defined characteristics,2,3 coupled with
the increasing demand for new tools in biotechnology, has resulted
in the development of NPs as platform for a variety of applications
including vaccine development, biological imaging as well as
targeted gene and drug delivery.4–7 Furthermore, there is also in-
creasing interest in the conjugation of functionally intact proteins
such as antibody fragments or viral proteins to nanoparticles,
enabling targeted delivery to specific cell types.8–10 Concurrently,
the immobilisation of enzymes on to NPs for applications in
biocatalysis has also been explored.11–13 Immobilisation of enzymes
on to NPs offers several operational advantages over other more
traditional supports. For example, NPs can be finely dispersed in
aqueous media thus reducing mass transfer problems associated
with enzymes immobilised on conventional bulk supports. They
can also be easily recovered and reused by centrifugation, or
magnetic sedimentation in the case of magnetic nanoparticles.11,14
However, all the reports of protein immobilisation on NPs
above rely either on non-covalent immobilisation methods (e.g.
hydrophobic adsorption) that are unstable to a wide range of ex-
perimental conditions, or non-specific reactions (e.g. succinimide
esters with the amino groups of exposed Lys residues) that result
in a heterogeneous population of bound proteins with random
Results and discussion
In order to immobilise the ybbR-tagged proteins, CoA–NP
conjugates were first prepared (Scheme 1). Starting from
carboxy-functionalised NPs (average diameter 191 nm), a long
polyethylene glycol spacer was introduced to the carboxy ter-
mini via in situ succinimide ester formation with 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDCI), to distance the
CoA sites from the bulk surface of the NP. Subsequently,
g-maleimidobutyric acid was coupled to the amino termini of
the appended PEG chains and any remaining unreacted amino
groups were capped with AcOSu. Michael addition of CoA
to the maleimide moiety furnished the desired CoA–NP. The
Sfp-mediated protein immobilisation was then attempted, ini-
tially with the ybbR-tagged model protein thioredoxin (Trx)15
and 12.5 mol% Sfp relative to the ybbR–Trx. Zeta potential
measurements of the nanoparticle suspension pre- and post-
reaction indicated a change from -40.8 1.1 mV to -34.1
1.7 mV for the CoA–NP and (ybbR–Trx)–Ppant–NP respec-
tively. In contrast, the control immobilisation reaction where
the Sfp was omitted gave no statistically significant change
School of Chemistry and Manchester Interdisciplinary Biocentre, The
University of Manchester, 131 Princess Street, Manchester, UK M1 7DN
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on biocatalysis.
‡ Electronic supplementary information (ESI) available: Additional
figures. See DOI: 10.1039/b916773k
§ E-mail: jason.micklefield@manchester.ac.uk or L.S.Wong@manchester.
ac.uk; Fax: +44 (0)161-306-8918; Tel: +44 (0)161-306-4509
(-39.8
0.4 mV). Preliminary tests to determine the enzy-
matic activity of an immobilised protein was also carried out
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Fig. 1 Strategy for the immobilisation of ybbR-protein fusions on to CoA-functionalised polystyrene nanoparticles mediated by Sfp.
Table 1 The decarboxylation kinetic constants for WT and ybbR-
fused enzymes with phenylmalonate as a substrate and m/z determined by
ES-MS
by the immobilisation of ybbR-tagged glutathione-S-transferase
(ybbR–GST) on to CoA–NPs, followed by detection of the enzyme
catalysed reaction of glutathione with 1-chloro-2,4-dinitrobenzene
(CDNB).15 This indicated that GST did indeed retain biocatalytic
activity post-immobilisation on to NPs (Fig. 2). In parallel control
experiments where Sfp was omitted from the immobilisation
reaction, only trace levels of GST activity were detected, which
probably arises from small amounts of non-specifically adsorbed
GST.
Enzyme
Km/mM
k
cat/s-1
k
cat/Km/mM-1s-1 m/z (Da)a
WT AMDase
ybbR–AMDase 9.9
WT GluR
ybbR–GluR
10.7 (26.9)b 316 (279)b 29.53 (10.37)b
25799 [25800]
27353 [27348]
29060 [29058]
30607 [30606]
358
36.16
0.08
3.1
1.8
0.24
44.51
25.01
a Value in brackets indicates calculated m/z for [M + H]+ b With 2-methyl-
2-phenylmalonate as substrate
To further demonstrate that Sfp-catalysed immobilisation of
functional proteins on to NPs can be achieved without the loss
of enzymatic activity, the immobilisation of two biocatalytically
interesting proteins, arylmalonate decarboxylase (AMDase)
enzyme from Bordetella bronchiseptica and glutamate racemase
(GluR) from Aquifex pyrofilus, was then attempted. The AMDase
catalyses a cofactor-independent and highly enantioselective
decarboxylation of arylmalonates, leading to valuable chiral
a-substituted acids.17,18 The GluR enzyme, in addition to its native
racemase activity,19 is also able to catalyse the decarboxylation of
phenylmalonate through a similar mechanism.18 This dual reactiv-
ity of GluR is noteworthy since the enzyme shows decarboxylase
activity at low temperatures and racemase activity above 55 ◦C.18,19
Accordingly, the optimised genes encoding AMDase and GluR
were cloned into an in house modified pET30b vector which
contains the ybbR sequence.15,18 Overexpression of these genes
in E. coli gave the desired proteins with an N-terminal ybbR-tag
and C-terminal His6-tag, allowing purification by Ni-affinity
chromatography. The purified proteins exhibited the desired mass
(by MS) and decarboxylase activity (Table 1). The kinetic data
indicate that in the case of the AMDase, the appended ybbR-tag
did not significantly alter enzyme activity. For GluR, notably
higher activity was observed with the ybbR-fused protein, which
may be due to improved stabilisation and solubility of the tagged
enzyme.
Subsequently, both ybbR–AMDase and ybbR–GluR were im-
mobilised on to the NPs under similar conditions to that of ybbR-
Trx and tested for decarboxylase activity. Where phenylmalonate
was used as the substrate, the Km values were 28.6 and 10.0 mM for
the immobilised ybbR–AMDase and –GluR respectively, approx-
imately three-fold higher than the wild-type enzymes indicating
weaker binding of the substrate. The immobilised enzymes also
exhibited lower turnover rates, with estimated kcat values of 1.70
and 0.01 s-1 for the ybbR–AMDase and –GluR respectively. Taken
together, the immobilised enzymes display a lower efficiency com-
pared to their counterparts in solution. This was thought to be due
to the electrostatic repulsion of the negatively charged substrate
attempting to approach the NPs that were also negatively charged
due to the unreacted surface carboxylates. Steric hindrance of the
immobilised enzymes may also play a role. However, calculation
of the exact kcat values was challenging since these were dependent
on the amount of enzyme immobilised, which was technically
difficult to determine due to the small amounts of protein
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Fig. 2 Graph of increase in CDNB–glutathione product concentration against time generated by the NPs after protein immobilisation reactions.
Scheme 1 Preparation of CoA-NP conjugates. Reagents and conditions: (a) EDCI, HOSu, MES buffer pH 6, 15 min; then bis-amino polyethylene glycol,
3 h; (b) GMBS, DMF, MES buffer pH 6, 3 h; then AcOSu, 1.5 h; (c) CoA, MES buffer pH 6, 2 h.
involved.¶ The results reported here are therefore likely to be an
underestimate of the true catalytic efficiency of the immobilised
enzymes. Nevertheless, the immobilised ybbR-AMDase was also
tested in decarboxylation of 2-methyl-2-phenylmalonate and its
conversion to (R)-2-phenyl-propionic acid occurred with 99% ee.18
The high enantioselectivity suggests that the AMDase does not
undergo denaturation during immobilisation and further suggests
that the enzyme is attached to the nanoparticles via the ybbR-tag.
With such a small enzyme, immobilisation via nonspecific surface
residues can result in a change in conformation that could affect
the enantioselectivity. The Km of the AMDase decarboxylation of
2-methyl-2-phenylmalonate was 66 mM, which is three-fold lower
compared with the free enzyme, in line with the trend above.
In order to study the stability and recyclability of the NP-
immobilised enzymes, the AMDase decarboxylase activity was
assayed repeatedly (Fig. 3). Here, it was found that the NP could
be recovered quantitatively by centrifugation and resuspension
prior to each assay cycle. Relative to the first reaction cycle (Fig. 3
and Fig. S1‡), the conversion decreased by 5% after the first
reuse (i.e. at cycle two), most probably due to the loss of non-
specifically adsorbed protein which was washed out. Only a non-
statistically significant 1% decrease was observed thereafter in all
further cycles, indicating the activity was maintained and stability
of the enzyme was good. For the NP where the immobilisation
was conducted in the absence of Sfp and therefore where only
¶ In order to calculate the kcat values, quantification of the amount of
protein immobilised was required. This was determined by the difference
in the amount of protein present in the reaction solutions pre- and
post-immobilisation with the both Bradford and BCA assays. Relatively
large values were returned: 86 or 15 mg per 100 mL suspension for ybbR–
AMDase and –GluR respectively. These were inconsistent with the zeta
potential measurements (which gave <10 mV change post-immobilisation)
and at or below the detection limit of FT-IR analysis (Fig. S2‡) of
<50 mg per 100 mL. There thus appears to be a systematic error in protein
quantification, which may be due to the presence of trace amounts of
nanoparticles present in the supernatant solutions after centrifugation as
well as interference from the Tween 20 in the protein solutions.
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Experimental
Materials and equipment
The PL-Latex Supercarboxyl White nanoparticles (batch no:
CML846W, charge density: 152.5 meq g-1) were donated by
Dr Simon Reid at Polymer Laboratories (Church Stretton,
UK). The bis-amino oligoethylene glycol of MWave 2000 Da
and CoA trilithium salt was purchased from Sigma-Aldrich
(Gillingham, UK) while g-maleimidobutyric acid succinimidyl
ester was purchased from TCI Europe (Zwijndrecht, Belgium).
The enzyme substrates glutathione, CNDB,20 D-glutamic acid
and phenylmalonic acid were purchased from Fisher Scientific
(Loughborough, UK). The 2-phenyl-2-methylmalonic acid18 and
AcOSu21 were synthesised according to previously reported proce-
dures. Sfp, ybbR-Trx and ybbR-GST were expressed and isolated
from E. coli as previously described.15,22
The zeta potential, a measure of the surface charge at the
interface between the particle and the solution, was measured with
a Zetasizer Nano from Malvern Instruments (Malvern, UK) with
the nanoparticles dispersed in 0.5 mM MES buffer with the values
determined over an average of three measurements at 20 runs
per measurement. The solid content of the NP suspensions was
determined by turbidometry, measuring the apparent absorbance
at 620 nm and comparison with a calibration curve of samples of
known amounts. Mass spectrometry of the proteins was performed
on a Micromass LCT electrospray time-of-flight mass spectrome-
ter (Waters, Milford, MA, USA). The FT-IR spectra were recorded
on a Tensor-27 FTIR spectrophotometer with a BioATR II unit
from Bruker Optics (Ettlingen, Germany) at the National Physical
Laboratory (Teddington, UK). All enzyme assays were performed
on an Anthos Zenyth 3100 96-well microplate reader.
Fig. 3 Graph of % reaction conversion of phenylmalonate to phenylacetic
acid by immobilised ybbR–AMDase over four reaction cycles where the
enzyme was immobilised in the presence of Sfp (blue bars) and with Sfp
omitted (red bars).
Table 2 The racemisation kinetic constants for WT, ybbR-fused and NP-
immobilised ybbR-fused GluR using D-glutamic acid as a substrate
Enzyme
Km/M
k
cat/s-1
k
cat/Km/M-1s-1
WT GluR
0.55 ¥ 10-4
0.44 ¥ 10-4
0.33 ¥ 10-4
0.056
0.060
101.8
136.3
ybbR-GluR
(ybbR–GluR)–Ppant–NPa
ca. 0.001a ca. 3.03a
a The value of kcat is likely to be an underestimate as accurate determination
of amount of enzyme immobilised is technically difficult to determine.‡
non-specifically bound proteins were present, the activity was more
than three-fold lower (Fig. 3). Moreover, a 20% decrease in relative
activity was observed after every cycle (Fig. S1‡). Thus only
specifically bound protein maintained high operational stability.
The immobilised ybbR–GluR was also assayed for racemase
activity with D-glutamic acid. The Km value for NP-immobilised
racemase was very similar to the one found for non-immobilised
enzyme (Table 2) although turnover rate was slower with the
estimated kcat value appearing 33-fold lower than the enzyme in so-
lution. However, the immobilised enzyme remained thermostable
and was able to perform the transformation at temperatures up to
55 ◦C.
Derivatisation of carboxy-nanoparticles with bis-amino
oligoethylene glycol
An aliquot of the supplied nanoparticle suspension (600 mL,
9.1 meq) was mixed with water (4248 mL), 0.5 M MES buffer,
pH 6.1 (600 mL) and HOSu solution (276 mL of 50 mg mL-1
solution). EDCI hydrochloride (19.2 mg, 0.1 mmol) was dissolved
in water (1 mL) and 276 mL of this was immediately added to the
nanoparticle mixture. The entire mixture was shaken for 15 min
then centrifuged at 7000 g for 5 min and the supernatant discarded.
The pellet was resuspended with 50 mM MES buffer, pH 6.0
(6 mL). The bis-amino oligoethylene glycol (90 mg, 45 mmol) was
dissolved in the same buffer (500 mL), added to the suspension
and the mixture shaken for 3 h. The suspension was centrifuged,
the supernatant discarded and the pellet resuspended as above.
Zeta potential measurements comparing the starting material and
the product (from -53.0 to -35.7 mV, 17.3 mV change) indicated a
33% ◦conversion. This suspension could be stored for several weeks
at 4 C for use in subsequent steps (where necessary, the stored
material was resuspended prior to use by shaking).
Conclusion
Overall, the immobilisation of model proteins and biocatalytically
relevant enzymes on to polystyrene nanoparticles using the site-
specific covalent ybbR-tag strategy under mild conditions was
demonstrated. These nanoparticles could be conveniently han-
dled and the immobilised enzymes retained their activity (albeit
somewhat lower than the enzymes in solution) over several rounds
of recycling, showing good working stability. Nevertheless, there
remains considerable further scope for the modulation of protein
activity through changing the surface chemistry of the NPs. It
should be noted that the immobilisation strategy employed here
is generic and could be applied to a wide range of other proteins
and nanoparticulate platforms such as magnetic nanoparticles for
targeting cancer cells.14
Derivatisation of amino oligoethylene glycol nanoparticles with
c-maleimidobutyric acid
A 2 mL aliquot of the amino-pegylated NP from the previous step
was mixed with a solution of g-maleimidobutyric acid succinimidyl
ester (GMBS, 11.2 mg dissolved in 100 mL DMF) and shaken for
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3 h. The remaining amino groups were then capped by addition
of AcOSu (28 mg dissolved in 100 mL DMF) to the mixture and
shaking for 1.5 h. The suspension was then centrifuged at 7000 g for
5 min and the supernatant discarded. The pellet was resuspended
with 50 mM MES buffer, pH 6.0 (2 mL). The centrifugation,
decanting of supernatant and resuspension was then repeated
once. This NP suspension was then used immediately.
pH 7.5 for the L-GDH racemase assay. The combined supernatants
were then assayed for protein content by the Bradford or BCA
assay and the amount of protein immobilised calculated by
deducting this value from the amount of protein added in the
immobilisation reaction solution above. The percentage solids in
the NP solution post-reaction were determined by turbidometry.
Assay of GST activity for immobilised ybbR-GST with CNDB
assay
Chemoligation of CoA to maleimido-functionalised nanoparticles
The maleimido-functionalised NP from above (1 mL) was mixed
with a solution of CoA (trilithium salt, 7.8 mg in 25 mL water) and
shaken for 2 h. The mixture was then centrifuged as above and
reconstituted with 50 mM HEPES, 50 mM NaCl buffer, pH 7.0
(750 mL). This suspension was used immediately.
A working solution of glutathione (7.5 mL of 100 mM aqueous
solution) and CNDB (7.5 mL of 100 mM in EtOH) mixed with
50 mM MES buffer pH 6.0 (to 90 mL) was prepared. Aliquots
of this working solution (50 mL) was added to the nanoparticle
suspensions (50 mL) in a 96-well plate, mixed and the absorbance
at 340 nm measured every 10 s for 5 min. The amount of
product formed was calculated from the absorbance and molar
absorption coefficient, adjusted to the background absorption of
the nanoparticle suspension without substrate.
Sub-cloning and expression of AMDase and GluR
Genes encoding AMDase from Bordetella bronchiseptica KU1201
and GluR from Aquifex pyrophilus were sub-cloned into modified
pET30b plasmid, containing ybbR sequence, by using HindIII and
XhoI restriction sites (see construct below).
Double-tagged proteins were expressed under standard condi-
tions in BL21(DE3) E. coli. 1% Triton X-100 was added during
lysis to maximise protein recovery. Purification was performed on
a GE Healthcare Bio-Science HiTrap Chelating column (5 mL)
using a step gradient of imidazole (25, 60 and 250 mM). This
procedure yielded typically 6–12 mg of protein per 500 mL of
culture and proteins were typically ca. 95% pure as determined by
SDS-PAGE.
Determination of decarboxylase kinetic constants for
non-immobilised AMDase and GluR by the Bromothymol Blue
(BTB) assay
Aqueous solutions of phenylmalonate or 2-methyl-2-
phenylmalonate (10 mL, 20-500 mM adjusted to pH 7.2)
were added to MOPS buffer (188–189 mL, 5–10 mM, pH 7.2)
containing Bromothymol Blue (0.0186 mM). The reaction was
initiated by addition of protein solution (1–2 mL) to a final enzyme
concentration of 0.002–0.02 mM. The change of absorbance of
BTB at 620 nm was then measured over time and the values of
Km and kcat were calculated as described previously.18
Sfp-mediated protein immobilisation on CoA-derivatised
nanoparticles
Determination of decarboxylase kinetic constants and conversion
for AMDase and GluR immobilised on nanoparticles by the
Bromothymol Blue (BTB) assay
An aliquot of the CoA-derivatised NP (500 mL) was centrifuged
as above and the supernatant discarded. Separately, the ybbR–Trx
(150 mL of 153 mM in 50 mM HEPES, 200 mM NaCl, pH 7.0)
or ybbR–GST (450 mL of 71 mM in 50 mM HEPES, 200 mM
NaCl, pH 7.0) or ybbR–AMDase (150 mL of 227 mM in 25 mM
TRIS, 0.2 mM DTT, pH 8.0) or ybbR–GluR (350 mL of 84 mM in
25 mM potassium phosphate buffer, pH 7.5) was mixed with Sfp
(495 mL of 62 mM in 50 mM HEPES, 200 mM NaCl, pH 7.0),
MgCl2–DTT stock solution (0.1 M MgCl2 and 0.5 M DTT,
3 mL per 500 mL of reaction mixture), Tween 20 stock solution
(25% v/v in 50 mM HEPES, 50 mM NaCl, sufficient to achieve a
2% v/v final concentration) and sufficient buffer (50 mM HEPES,
50 mM NaCl, pH 7.0) to make up the volume to 1 mL. 500 mL
of this protein reaction solution was then used to reconstitute the
NP pellet and the suspension incubated at 37 ◦C for 1 h. The
suspension was then precipitated by centrifugation as above, the
supernatant decanted and the pellet was then resuspended with
25 mM phosphate buffer with 2% v/v Tween 20 (500 mL). This was
repeated once, then the pellet was resuspended in the appropriate
buffer (500 mL) for the enzyme assays below, either 10 mM MOPS,
pH 7.2 for the BTB decarboxylation assay or 25 mM TRIS buffer
TRIS buffer (1000 mL, 0.1 M, pH 8) was added to an aqueous
solution of phenylmalonate (10, 20, 30, 40 mL, 0.5 M, pH 8) or
2-methyl-2-phenylmalonate (20, 30, 40, 50 mL, 0.5 M, pH 8).
Aqueous suspension of NP-immobilised enzyme (20 mL) was
added and the reaction was stirred vigorously at 30 ◦C for
given time periods then quenched with addition of aqueous HCl
(1.0 M to pH 4). The products were extracted with diethyl ether,
evaporated under reduced pressure and conversion was analyzed
by 1H NMR. The values of Km were calculated using a Lineweaver–
Burk plot.
Determination of glutamate racemase kinetic constants GluR and
GluR immobilised on nanoparticles by the L-glutamate
dehydrogenase (L-GDH) assay
A 50 ml of solution from GluR reaction and 10 ml of NAD+
(125 mM) were added to TRIS buffer (140 ml, 100 mM, pH 8).
The reaction was initiated by addition of 2 ml of L-GDH solution
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(15 mg per 500 ml) and the formation of NADH was determined by
measurement of absorbance at 340 nm. The velocity of the reaction
was compared with the velocities determined during calibration
with known amounts of L-glutamic acid. After calculation of
conversion levels, the value of Km was determined using a
Lineweaver–Burk plot.
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Acknowledgements
This work is supported by the EPSRC (Basic technology pro-
gramme EP/C523857/1 and LSI fellowship EP/F042590/1 for
LSW) and CoEBio3. The nanoparticles were donated by Dr Simon
Reid (PolymerLabs). Assistance with zeta potential measurements
by Prof. Stephen Yeates (Manchester), FT-IR spectroscopy by Dr
Alex Knight (National Physical Laboratory) are also gratefully
acknowledged.
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