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Journal of Environmental Biology ISSN: 0254- 8704
CODEN: JEBIDP

Optimization of the whole-cell catalytic activity of

recombinant Escherichia coli cells with surface-immobilized
organophosphorus hydrolase
Hongxing Zhang, Qianqian Li, Ting Ye, Zhen Zhang and Lin Li*
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology,
Huazhong Agricultural University, Wuhan, 430070, China
*Corresponding Author email : lilin@mail.hzau.edu.cn

Abstract
Previous studies have verified the feasibility of using Escherichia coli systems that display
organophosphorous hydrolase (OPH) on the cell surface as whole­cell catalysts. However, the
inefficient display of the enzyme on cell surfaces remains unaddressed. In the present study, multiple
Publication Info optimization experiments on full­length and truncated ice nucleation protein anchors, E. coli host
cells, culture media, and culture conditions were performed to optimize whole­cell OPH enzymatic
Paper received: activity. The results show that apart from the dramatic effect of isopropyl­E­ D ­thiogalactoside
20 June 2012 concentration and culture temperature, the coordination between the anchor protein, culture
media, and host cells is essential for highly efficient OPH display. Under optimal conditions, namely,
Revised received: culturing in M9 medium, 20 °C induction temperature, 0.1 mmol l­1 IPTG, and 100 μmol l­1 Co2+, the
12 August 2012 engineered E. coli strain MB109­406 that expresses the fusion enzyme InaK­N­OPH exhibited a whole­
cell OPH activity of 0.62 U mg­1 × cell d.wt. This result is much higher than that of several currently
Accepted: available OPH­displaying systems, which shows the potential of the current system for further large­
scale industrial or environmental applications.
29 August 2012
Key words
Cell surface display, Organophosphorus hydrolase, Optimization, Ice nucleation protein,
Escherichia coli

Introduction Organophosphorus hydrolase (OPH; EC 3.1.8.1) is

The long-term practice of controlling agricultural
pests using various organophosphate (OP) pesticides in
China has resulted in serious toxic residues, which
constitutes a significant environmental threat. The biological
process based on microbial phosphotriesterases is a feasible
solution for detoxifying such OP agents, including several
synthetic, neurotoxic substances. Among various
previously developed biological technologies, the bacterial
cell surface display strategy is of particular interest in the
development of the whole-cell catalysts by immobilizing
and functionalizing OP-degrading enzymes onto the surface
of host cells, which promotes reaction rates and overcomes
the mass transfer limitations of macromolecules and toxic
substrates (Daugherty, 2007; Lee et al., 2003).
a homodimeric phosphotriesterase isolated from
Pseudomonas diminuta (McDaniel et al., 1988) and
Flavobacterium sp. (Mulbry and Karns, 1989). This
enzyme detoxifies a broad range of OP-like agents by
hydrolyzing various phosphorus ester bonds including P -
O, P - F, P - CN, and P - S when a metal ion exists as a
cofactor for its activity (Grimsley et al., 1997; Lai et al.,
1995). The heterologous intracellular expression of these
OPHs in Escherichia coli systems may allow the
development of large-scale detoxification processes because
they can be synthesized at relatively higher levels compared
with their original hosts (Chen and Mulchandani, 1998).
However, the limited substrate diffusion of directly using
recombinant cells caused by cell membrane barriers need to be

© Triveni Enterprises, Lucknow (India ) Journal of Environmental Biology, Vol. 34, 315-319, April 2013

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316
considered. As an alternative approach to address this
problem, several previous studies displayed OPH on the
surface of various bacteria using different anchoring
proteins. These studies which include the Lpp-OmpA
anchor in E. coli (Richins et al., 1997), GPI anchor in
Saccharomyces cerevisiae (Takayama et al., 2006), and ice
nucleation protein (INP) from Pseudomonas syringae in E.
coli (Cho et al., 2002; Shimazu et al., 2001), and Pseudomonas
putida (Lei et al., 2005; Shimazu et al., 2003) have
demonstrated the feasibility of these approaches.
The INP-anchored system in Gram-negative bacteria
has been used most often because of the broad availability
of this anchor. Previous investigations verified that only
partial intracellular enzymes could be expressed on cell
surfaces, resulting in the limited degradation activity of
whole-cell catalysts (Li et al., 2004; Li et al., 2011). Therefore,
the development of more efficient INP-mediated display
systems remains critical. In the present study, the anchoring
activities of full-length and truncated INP proteins were
compared to maximize the detoxifying activity of surface-
immobilized OPH. In addition, the E. coli host cells and
culture media were screened for better expression and
surface-immobilization of the fused OPH enzymes.
Subsequently, an orthogonal trial was performed to optimize
IPTG induction and culture conditions to maximize the whole-
cell OPH activity of the engineered E. coli whole-cell catalyst.
The effect of storage time on the whole-cell OPH activity of
the engineered strain was also investigated.
Materials and Methods
Strains, plasmids and culture conditions : E. coli DH5α
was used to construct the recombinant plasmids. E. coli
JM109, W3110 and BL21(DE3) were used as host strains for
OPH expression and surface display. The wild-type P.
syringae KCTC1832 provided the inaK gene that encodes
the ice nucleation protein InaK. The wild-type
Flavobacterium sp. ATCC27551 provided the gene opd that
encodes OPH. The recombinant plasmids pMB405, pMB406,
and pMB407, which respectively harbor the fusion gene
with inaK-opd, inaK-N-opd, and inaK-NC-opd were
constructed for the expression and display of the
corresponding proteins.
All E. coli strains were grown in a Luria-Bertani
medium (LB) unless specified otherwise. The recombinant
E. coli strains were cultured at 37 °C in M9, GYT and SOC
media (Sambrook and Russell, 2001) containing ampicillin
at a final concentration of 50 mg ml-1 for OPH expression
and surface display. To induce the expression of the target
proteins in E. coli, isopropyl-E-D-thiogalactoside (IPTG)
was added at final concentrations ranging from 0.1 mmol l-1
to 0.3 mmol l-1 when the OD600 reached 0.4.
Journal of Environmental Biology, April 2013
H. Zhang et al.
Plasmid construction and transformation : Gene inaK was
PCR-amplified from the P. syringae KCTC1832 genome with
the primers F inaK : 5¢-TGCTGCCATGGCTC TCGAC
AAGGCGTTGG-3¢ (NcoI site underlined) and RinaK: 5¢-GAA
GATCTTACCTCTATCCAGTCATCGTCCTCG-3¢ (BglII site
underlined), and the opd gene was PCR-amplified from the
Flavobacterium ATCC27551 genome (GenBank accession
no. M29593.1) with the primers F opd: 5¢-
GGAGATCTATCGGCACAGGCGATCGG-3¢ (BglII site
underlined) and Ropd: 5¢-GGAAGCTTTC ATGACGCCCG
CAAGGTCG-3¢ (HindIII site underlined) to construct the
recombinant plasmid pMB405. The two amplified products
were digested with NcoI/BglII, and BglII/HindIII,
respectively, and then inserted consecutively into the NcoI/
BglII and BglII/HindIII sites of the E. coli expression vector
plasmid pTrcHis-C (Invitrogen) to create the pMB405 plasmid
(8723 bp). Similarly, truncated inaK-N was PCR-amplified
with the primers FinaK and RinaK-N: 5¢-TAAGATCTGGT
CTGCAAATTATTCTGCGGCGTCGTCACCGG-3¢ (BglII
site underlined), and then inserted into the NcoI/BglII sites
of pMB405 to yield the pMB406 plasmid (5819 bp). The
primers FinaK-C: 5¢-TTCAGATCTGGGACGG GAAGA
GGTAC-3¢ (BglII site underlined) and Ropd were used to
amplify the inaK-C-opd gene fragment from pMB405 via
PCR. The amplified fragment was digested with BglII/HindIII
and inserted into the BglII/HindIII sites of pMB406, resulting
in the recombinant plasmid pMB407 (5963 bp).
The transformation of E. coli was performed using
standard procedures (Sambrook and Russell, 2001). The
transformed E. coli JM109 strains that harbored pMB405,
pMB406 and pMB407 were designated as MB109-405,
MB109-406 and MB109-407, respectively.
SDS-PAGE : Fusion proteins prepared from the whole-cell
samples were analyzed via 12% SDS-PAGE using standard
procedures (Sambrook and Russell, 2001).
Measurement of whole-cell OPH activity : Paraoxon-ethyl
(Dr. Ehrenstorfer GmbH, Germany) with 95.5% purity was
used as the substrate to measure the whole-cell OPH activity
following previously described procedures (Li et al., 2004).
One unit of OPH activity was defined as 1 μmol l-1 paraoxon-
ethyl hydrolyzed min-1 mg-1 of the cell d.wt..
Proteinase K accessibility assay : The proteinase K
accessibility assay was performed following the previously
described method (Richins et al., 1997). The cells (1 ml,
OD600 = 1.0) were centrifuged and resuspended in 1 ml of
proteinase K solution with 15% (w/v) sucrose, 15 mM Tris-
HCl, 0.1 mM EDTA (pH 7.8) and 200 mg proteinase K (Sigma-
Aldrich). The cells were then incubated at 37°C for 1 hr
following the whole-cell OPH activity measurement as
described above.
Optimization of E. coli surface-immobilized OPH activity 317

Statistical analysis : All data were the average of triplicate
assays. Statistical analysis was carried out using the SPSS
18.0 statistical software. Differences with p-values less than
0.05 were considered statistically significant.
Results and Discussion
Construction and expression of the InaK-OPH, InaK-N-
OPH, and InaK-NC-OPH fusion proteins : The recombinant
plasmids pMB405, pMB406 and pMB407, which encode
InaK-OPH, InaK-N-OPH and InaK-NC-OPH, respectively,
were constructed to create binary and tripartite fusion genes
in a single whole encoding frame driven by the trc promoter
upon IPTG induction. These plasmids were then introduced
into E. coli JM109 to generate the transformed strains
MB109-405, MB109-406 and MB109-407.
All transformed strains were subjected to whole-cell
OPH activity assays after IPTG (0.2 mmol l-1) induction and
shake-flask incubation for 6 hr. Unlike the limited whole-cell
OPH activity of E. coli constructs that express intracellular
OPH, all of the transformed strains that harbored the fused
opd gene exhibited substantial whole-cell OPH activity. This
result indicates that fusion enzymes were immobilized onto
the surface of the host cells by the full-length and the
truncated InaK anchors during active enzyme confirmation.
Interestingly, the transformed strain MB109-406 that
expressed InaK-N-OPH exhibited a maximum activity of 0.46
U mg-1 d.wt., which was comparable to the InaK-NC-OPH
expression in the transformed strain MB109-405 (by 0.44 U
mg-1 d.wt.), but much higher than the InaK-OPH expression
in the transformed strain MB109-405 (0.28 U mg-1 d.wt.).
These results suggest that InaK-N is a more efficient anchor
than InaK-NC and InaK because of its smaller molecular
size and higher anchoring efficiency for displaying OPH.
Therefore, the InaK-N–mediated OPH display system was
chosen for further analysis.
InaK-N-OPH expression was detected by SDS-PAGE
(Fig. 1a), which showed that the InaK-N-OPH fusion protein
was synthesized with the predicted molecular mass (~57
kDa). The full wavelength (from 250 to 800 nm) absorption
spectrum of the MB109-406–catalyzed reaction solution was
recorded (Fig. 1b); given the maximum spectral peak of the
reaction products at 400 nm, which is the distinct adsorption
peak of p-nitrophenol (Gao et al., 2011) as a product of
paraoxon-degrading reaction, the activity of surface-
displayed InaK-N-OPH was confirmed.
A proteinase K accessibility assay was performed
to verify the surface localization of InaK-N-OPH in the
transformed MB109-406 cells. Considering intracellular
InaK-N-OPH is inaccessible to externally added proteinase
K, the presence of the surface-displayed fusion enzyme
could easily be determined by analyzing whole-cell OPH
activity. As shown in Fig. 2, the MB109-406 whole-cell
activity decreased by about 91% after proteinase K treatment
compared with only a slight reduction in the control strain
that expresses intracellular OPH. Therefore, the fusion
enzyme was anchored successfully onto the outer membrane
of the MB109-406 cells.

a b
5

2 (i)
( ii)

1
(iii)
( iv )
0
2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 5 5 0 6 0 0 6 5 0 70 0 7 5 0 8 0 0
W a vele n gth (n m )

Fig. 1 : SDS-PAGE analysis of E. coli MB109-406 whole-cell proteins (a) and full wavelength adsorption spectra of the paraoxon degradation
products by E. coli MB109-406 (b). (a) lane M, protein MW marker; lane 1, E. coli JM109; lane 2, MB109-406 without IPTG induction; lane
4, MB109-406 with IPTG induction. (b) MB109-405, the distinct adsorption peaks at 400 nm indicate the occurrence of p-nitrophenol, a
product of paraoxon degradation. (i) purified OPH; (ii) MB109-406; (iii) without paraoxon; (iv) without MB109-406

Journal of Environmental Biology, April 2013

318
Fig. 2 : Whole-cell OPH activity assay using proteinase K treatment.
(A) E. coli MB109-406; (B) transformed E. coli strain that expresses
cellular OPH. Analyses were based on unit cell density (OD600 = 1).
Values are mean of three replicates + SD
Experimental groups
Fig. 3 Summarized orthogonal trial (L9, 3 4) optimization of the E.
coli MB109-406 culture conditions. Experimental group I: 10 °C,
0.1 mmol/l IPTG, 4 h, 50 mmol/l Co2+. Experimental group II: 10 °C,
0.2 mmol/l IPTG, 6 h, 100 mmol/l Co2+. Experimental group III: 10
C, 0.3 mmol/l IPTG, 8 h, 150 mmol/l Co2+. Experimental group IV:
15 °C, 0.1 mmol/l IPTG, 6 h, 150 mmol/l Co2+. Experimental group
V: 15 °C, 0.2 mmol/l IPTG, 8 h, 50 mmol/l Co2+. Experimental group
VI: 15 °C, 0.3 mmol/l IPTG, 4 h, 100 mmol/l Co2+. Experimental
group VII: 20 °C, 0.1 mmol/l IPTG, 8 h, 100 mmol/l Co 2+ .
Experimental group VIII: 20 °C, 0.2 mmol/l IPTG, 4 h, 150 mmol/
l Co2+. Experimental group IX: 20 °C, 0.3 mmol/l IPTG, 6 h, 50
mmol/l Co2+
Time (days)
Fig. 4 : Time course of E. coli MB109-406 whole-cell OPH activity.
The cells were cultured in PBS buffer (pH 7.0) at 4 °C, and then
harvested for OPH activity determination. The analyses were based
on unit cell density (OD600 = 1)
Journal of Environmental Biology, April 2013
H. Zhang et al.
Effect of different E. coli hosts and culture media on the
activity of the surface-displayed InaK-N-OPH : The whole-
cell OPH activities of the three E. coli recipient strains JM109,
BL21(DE3), and W3110 were compared. In addition, the
effects of culturing the transformed E. coli cells in LB, M9,
GYT, and SOC on InaK-N-OPH expression were determined
by comparing their whole-cell OPH activity. Among the
various combinations with E. coli hosts and culture media,
E. coli MB109-406 that expresses InaK-N-OPH was
confirmed to exhibit the highest whole-cell OPH activity
during culturing in M9 medium.
Optimizing whole-cell activity with orthogonal trials : The
single factors IPTG concentration, IPTG induction time,
culture temperature, and Co2+ concentration significantly
affected the whole-cell OPH activity. Based on these results,
L9 (34) orthogonal trial was designed to optimize these
factors at the following levels: 0.1, 0.2 and 0.3 mmol l-1 IPTG;
4, 6 and 8 hrs induction times: 10, 15 and 20°C culture
temperature and 50, 100 and 150 mmol l-1 Co2+. The results
indicate that the maximum whole-cell OPH activity (0.62 U
mg-1 cell d.wt.) was obtained when MB109-406 was
incubated at 20 °C for 8 hr with 0.1 mmol l-1 IPTG and the
final Co2+ concentration was maintained at 100 μ mol l-1 (Fig.
3, experimental group 7). This optimized whole-cell OPH
level was much higher than those observed in several
previously reported OPH-displaying systems: E. coli XL1-
blue displaying OPH with an InaV INPNC anchor (Shimazu
et al., 2001) by 13.7-fold; E. coli XL1-blue displaying OPH
with Lpp-OmpA anchor (Richins et al., 1997) by 16.4-fold;
and E. coli XL1-blue displaying OPH with InaK-NC anchor
(Shimazu et al., 2001) by 80.2-fold. Therefore, the current
OPH-displaying system is an improved OP-degrading
whole-cell catalyst.
Stability of MB109-406 in maintaining whole-cell OPH
activity : Pesticide detoxification using recombinant cells in
large-scale immobilized bioreactors requires the production
of stable and active cultures without antibiotic supplements.
The stability of introduced pMB406 in MB109-406 under
ampicillin-free culture conditions was investigated in
successive inoculation at 12 hr intervals. The result shows
that about 60% of the MB109-406 cultures maintained the
plasmid pMB406 during the 7 day time course. In addition,
the whole-cell OPH activity during the 30 day storage of
MB109-406 at 4 °C showed a slowly decreasing pattern of
enzymatic activity, as shown in Fig. 4. The MB109-406 strain
retained 93% of its original activity over the 30 day
experimental time course, which indicates the great stability
of the surface-display system and its potential for further
large-scale or continuous operations.
A stable and regenerable cell platform is conducive
to maintain the activity of surface-displayed enzymes to
develop a successful surface-display system. Considering
Optimization of E. coli surface-immobilized OPH activity
coordinated cellular transcription and secretion is required
for the efficient surface display of heterologous proteins,
the introduction of the pMB406 plasmid into other E. coli
recipients such as BL21(DE3) and W3110 resulted in low
cell viability or low whole-cell OPH activity. Moreover, the
significant increase in whole-cell OPH activity when the
cultures were grown at relatively low temperatures (below
20 °C) indicate that temperature is crucial for maximum OPH
display. Therefore, this study opens new perspectives into
the development of more efficient whole-cell catalysts for
industrial and environmental applications.
Acknowledgment
This work was supported by the grant received from
the National Natural Science Foundation of China (Grant
nos. 31070111 and 30670054).
References
Chen, W. and A. Mulchandani: The use of live biocatalysts for pesticide
detoxification. Trends Biotechnol., 16, 71-76 (1998).
Cho, C.M., A. Mulchandani and W. Chen: Bacterial cell surface display
of organophosphorus hydrolase for selective screening of
improved hydrolysis of organophosphate nerve agents. Appl.
Environ. Microbiol., 68, 2026-30 (2002).
Daugherty, P.S.: Protein engineering with bacterial display. Curr.
Opin. Struct. Biol., 17, 474-80 (2007).
Gao, L.L., Y. Kunahong, L. Wei, D.W. Wang, D.H. Wang and F.X.
Gan: Measurement of p-nitrophenol with normalizationally
modified UV-Vis spectrum methodology in wide pH range
wastewater. Environ. Sci. Technol. China, 34, 111-114 (2011).
Grimsley, J.K., J.M. Scholtz, C.N. Pace and J.R. Wild: Organo
phosphorus hydrolase is a remarkably stable enzyme that
unfolds through a homodimeric intermediate. Biochemistry,
36, 14366-14374 (1997).
Lai, K., N.J. Stolowich and J.R. Wild: Characterization of P-S bond
319
hydrolysis in organophosphorothioate pesticides by
organophosphorus hydrolase. Arch. Biochem. Biophys., 318,
59-64 (1995).
Lee, S.Y., J.H. Choi and Z. Xu: Microbial cell-surface display. Trends
Biotechnol., 21, 45-52 (2003).
Lei, Y., A. Mulchandani and W. Chen: Improved degradation of
organophosphorus nerve agents and p-nitrophenol by
Pseudomonas putida JS444 with surface-expressed organo
phosphorus hydrolase. Biotechnol. Prog., 21, 678-681 (2005).
Li, L., D.G. Kang and H.J. Cha: Functional display of foreign protein
on surface of Escherichia coli using N-terminal domain of ice
nucleation protein. Biotechnol. Bioeng., 85, 214-221 (2004).
Li, Q., H. Ni, S. Meng, Y. He, Z. Yu and L. Li: Suppressing Erwinia
caratovora pathogenicity by projecting N-acyl homoserine
lactonase onto the surface of Pseudomonas putida cells. J.
Microbiol. Biotechnol., 21, 1330-1335 (2011).
McDaniel, C.S., L.L. Harper and J.R. Wild: Cloning and sequencing
of a plasmid-borne gene (opd) encoding a phosphotriesterase.
J. Bacteriol., 170, 2306-2311 (1988).
Mulbry, W.W. and J.S. Karns : Parathion hydrolase specified by the
Flavobacterium opd gene: relationship between the gene and
protein. J. Bacteriol., 171, 6740-6746 (1989).
Richins, R.D., I. Kaneva, A. Mulchandani and W. Chen: Biodegradation
of organophosphorus pesticides by surface-expressed
organophosphorus hydrolase. Nat. Biotechnol., 15, 984-987
(1997).
Sambrook, J. and D.W. Russell: Molecular cloning: A laboratory
manual. 3rd Edn., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY (2001).
Shimazu, M., A. Mulchandani and W. Chen: Cell surface display of
organophosphorus hydrolase using ice nucleation protein.
Biotechnol. Prog., 17, 76-80 (2001).
Shimazu, M., A. Nguyen, A. Mulchandani and W. Chen: Cell surface
display of organophosphorus hydrolase in Pseudomonas putida
using an ice-nucleation protein anchor. Biotechnol. Prog., 19,
1612-1614 (2003).
Takayama, K., S. Suye, K. Kuroda, M. Ueda, T. Kitaguchi, K.
Tsuchiyama, T. Fukuda, W. Chen, and A.Mulchandani: Surface
display of organophosphorus hydrolase on Saccharomyces
cerevisiae. Biotechnol. Prog., 22, 939-943 (2006).
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