Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Expression of recombinant organophosphorus

hydrolase in the original producer of the enzyme,
Sphingobium fuliginis ATCC 27551

Kosuke Nakayama, Takeshi Ohmori, Satoshi Ishikawa, Natsumi Iwata, Yasuo

Seto & Kazuyoshi Kawahara

To cite this article: Kosuke Nakayama, Takeshi Ohmori, Satoshi Ishikawa, Natsumi Iwata, Yasuo
Seto & Kazuyoshi Kawahara (2016): Expression of recombinant organophosphorus hydrolase
in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551, Bioscience,
Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1123606

To link to this article: http://dx.doi.org/10.1080/09168451.2015.1123606

Published online: 19 Jan 2016.

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 Bioscience, Biotechnology, and Biochemistry, 2016
Note
Expression of recombinant organophosphorus hydrolase in the original
producer of the enzyme, Sphingobium fuliginis ATCC 27551
Kosuke Nakayama1, Takeshi Ohmori2, Satoshi Ishikawa1, Natsumi Iwata1, Yasuo Seto2 and
Kazuyoshi Kawahara2,*
1
Department of Biosciences, College of Science and Engineering, Kanto Gakuin University, Yokohama, Japan;
2
Third Department of Forensic Science, National Research Institute of Police Science, Kashiwa, Japan
Received September 16, 2015; accepted November 13, 2015
http://dx.doi.org/10.1080/09168451.2015.1123606
The plasmid encoding His-tagged organophospho-
rus hydrolase (OPH) cloned from Sphingobium
Downloaded by [Umeå University Library] at 05:22 06 February 2016
fuliginis was modified to be transferred back to this
bacterium. The replication function of S. amiense
plasmid was inserted at downstream of OPH gene,
and S. fuliginis was transformed with this plasmid.
The transformant produced larger amount of active
OPH with His-tag than E. coli.
Key words: organophosphorus hydrolase; nerve
agent; paraoxon; Sphingobium amiense;
Sphingobium fuliginis
The species belonging to the family Sphingomon-
adaceae1) have unique and useful characteristics. The
bacteria have hydrophobic cell surface because the sur-
face membrane is composed of glycosphingolipids with
short sugar chain instead of lipopolysaccharides.2)
Many of the family members have been reported to
have degradation activity toward various xenobiotic
and recalcitrant compounds3) including dibenzo-p-
dioxin,4) gamma-hexachlorocyclohexane,5) and lignin.6)
The bacterium isolated as a degrader of organophos-
phorus pesticides from rice paddy of the Philippines7)
was first reported to belong to the genus Flavobac-
terium (Flavobacterium sp. ATCC 27551), but we iden-
tified the bacterium as Sphingobium fuliginis.8) In the
previous studies of many groups including ourselves,9–
11)
the enzyme that can hydrolyze organophosphorus
compounds, organophosphorus hydrolase (OPH), was
cloned in E. coli and genetically engineered to enhance
the activity. Since OPH was proved to exhibit the
hydrolytic activity toward organophosphorus nerve
agents such as sarin (O-isopropyl methylphosphonoflu-
oridate) and VX (O-ethyl-S-(2-diisopropylaminoethyl)
methylphosphonothiolate), it was expected to be
applied to the decontamination of these nerve agents in
the case of chemical terrorism, and the degrading activ-
ity toward VX has been improved.12–14) We also con-
structed the mutant OPH for the degradation of VX15)
and established the purification method using the
*Corresponding author. Email: kawahara@kanto-gakuin.ac.jp
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry
affinity between His-hexamer attached at N-terminal of
OPH and Ni-chelating resin. The mutant OPH was fur-
ther immobilized with porous silica or calcium alginate
to use it as a bioreactor (submitted for publication).
However, the cloned OPH easily formed insoluble
aggregate in E. coli, and the yield could not be easily
improved. Therefore, more efficient method of produc-
tion was required.
As Sphingobium spp. and E. coli are taxonomically
widely separated, genes of Sphingobium were assumed
to be efficiently expressed in Sphingobium cells than in
E. coli. But the plasmid constructed in E. coli could
not be transferred to Sphingobium spp. because we
used pTV118N (Takara Bio, Ohtsu, Japan), a derivative
of pUC118, as a cloning vector. As for the transforma-
tion of Sphingobium spp., the shuttle vector plasmid
between species of Sphingomonadaceae and E. coli
was reported by Saito et al.16) They showed that ORF4
of a plasmid of Sphingobium amiense was able to con-
fer the stability of the plasmid in Sphingobium and
other species of Sphingomonadaceae. Based on this
knowledge, we cloned the 2.9 kb ORF4 region and
inserted it to the HindIII site at 3’-end of OPH gene,
which was present between NcoI and HindIII sites of
pTV118N. The constructed plasmid, pTV118N-His-
OPH-ORF4, was used for the transformation of S.
fuliginis ATCC 27551, and the transformant was desig-
nated KGU0379. Additionally, Sphingomonas paucimo-
bilis KGU0001 (originally IAM 12576T) was also
transformed with the same plasmid, and the transfor-
mant was designated KGU0400. The transformants of
S. paucimobilis and S. fuliginis were disrupted by soni-
cation to prepare crude enzyme solution, and they were
purified by Ni-chelating resin. The OPH activity was
measured by the method described previously15) using
paraoxon (O,O-Diethyl p-nitrophenylphosphate) as a
substrate, and specific activity was calculated. As
shown in Table 1, both transformants of S. paucimo-
bilis and S. fuliginis showed clear activity of OPH, and
the specific activity of their crude enzyme preparations
are higher than that of E. coli stain KGU0255 harbor-
ing the same plasmid. All of purified preparations were
 2 K. Nakayama et al.
Table 1. Paraoxon-hydrolyzing activity of OPH produced by the bacteria harboring OPH-encoding plasmid.

Specific activity of OPH (U/mg)a)

Bacterial strain Recombinantplasmid Wild-typeOPH gene Crude enzyme Purified enzyme

1
E. coli KGU0255 present absent 0.6 × 10 1.1 × 101
S. paucimobilis KGU0400 present absent 1.8 × 101 0.6 × 101
S. paucimobilis KGU0001 absent absent 0 –
S. fuliginis KGU0379 present present 2.5 × 102 1.2 × 102
S. fuliginis ATCC 27551 absent present 1.4 × 102 1.2 × 101
a)
: His-tagged OPH was partially purified by Ni-chelating resin.

found to have specific activities similar to or lower than
those of crude ones, suggesting that the recombinant
OPH was partially inactivated during purification by
the unknown reason. The wild-type strain of S. fuliginis
also showed the activity, because this strain produces
native OPH, but the weak activity of the purified frac-
tion was deduced to be caused by non-specific binding.
Downloaded by [Umeå University Library] at 05:22 06 February 2016
around 39 kDa, and the molecular size fitted to the
calculated molecular weight (39,017 Da).
In the next experiment, degradation activity of crude
enzyme from S. fuliginis KGU0379 toward nerve
agents was measured as described previously.15) The
organophosphorus nerve agents, tabun (O-ethyl N,N-
dimethylaminophosphonocyanidate), sarin, cyclohexyl-

The crude and purified preparations of S. fuliginis
transformant, KGU0379, was submitted to Western blot
analysis to ensure that this strain could produce the
recombinant OPH with His-tag at N-terminal. His-
tagged protein was detected using anti-His-tag mono-
clonal antibody and secondary antibody, goat anti-
mouse IgG alkaline phosphatase conjugate (Novagen).
As shown in Fig. 1(B), His-tagged OPH was detected
both in crude (lane 2) and purified (lane 6) fractions.
The same band was also detected in the purified
fraction from E. coli KGU0255 (lane 7). The corre-
sponding protein bands for His-tagged OPH were
observed in SDS-PAGE (Fig. 1(A), lanes 6 and 7) at
sarin (O-cyclohexyl methylphosphonofluoridate), VX,
and RVX (O-isobutyl-S-(2-diethylaminoethyl)
methylphosphonothiolate) were synthesized in the labo-
ratory of National Research Institute of Police Science.
The synthesis and usage of the nerve agents were con-
ducted with the approval of the Ministry of Economy,
Trade, and Industry of Japan. As shown in Table 2, the
residual amounts of all of nerve agents including VX
and RVX were reduced by 20-min incubation com-
pared with control experiments, indicating that OPH
produced by S. fuliginis exhibited the degradation
activity toward nerve agents. The amounts of tabun,
sarin, cyclohexylsarin, and soman were reduced even

Fig. 1. Detection of His-tagged OPH by Western blotting.

Notes: OPH produced by S. fuliginis KGU0379 was purified by Ni-chelating column, and crude preparation and eluates from the column were
separated by SDS-PAGE, blotted, and treated with the primary antibody against His-hexamer and secondary antibody conjugated with alkaline
phosphatase. Lanes 1, molecular-weight marker (Low range marker, Bio-rad, Hercules, CA, USA); 2, crude preparation; 3, flow-through of crude
preparation; 4, eluate of binding buffer; 5, eluate of 60 mM imidazole; 6, eluate of 1 M imidazole (purified fraction); 7, purified fraction of OPH
produced by E. coli KGU0255. A, CBB staining of SDS-PAGE; B, antibody staining on PVDF membrane.

Table 2. Degradation of nerve agents by crude preparation of OPH from S. fuliginis KGU0379.

Residual ratio (%) of nerve agenta

Incubation Tabun Sarin Cyclo-hexylsarin Soman VX RVX

Solution
Without OPH 58.1 ± 7.0 59.3 ± 2.4 62.2 ± 4.9 80.7 ± 4.6 107.3 ± 9.1 104.7 ± 4.0
With OPH 0 0 1.7 ± 0.2 7.1 ± 0.5 57.1 ± 1.8 51.0 ± 1.7
Calcium alginate gel
Without OPH 5.7 ± 1.1 14.8 ± 1.1 12.7 ± 1.3 23.4 ± 1.9 49.1 ± 3.4 43.9 ± 3.7
With OPH 0 0 1.0 ± 0.3 2.3 ± 0.6 30.3 ± 3.1 22.9 ± 2.1
a
: Data of triplicated experiments were shown as mean ± SD.
 Expression of recombinant OP hydrolase in S. fuliginis
in the control experiments. These compounds are rather
unstable and deduced to be degraded nonenzymatically.
For the immobilization of OPH, sodium alginate was
added to the crude preparation of OPH to the final con-
centration of 2% and dropped to 2% calcium chloride
to form the gel of calcium alginate. The nerve agents
were also degraded in 20 min, although the recovery of
the nerve agents in the control experiments without
OPH was low probably because of the nonenzymatic
degradation and partial absorption to the gel (Table 2).
These results suggested that the solution or the immo-
bilized form of OPH can be used as a bioreactor to
degrade nerve agents in the case of chemical ter-
rorisms, for which we have developed effective on-site
technologies of detection and decontamination.17)
S. fuliginis grows rapidly, can be cultivated in large
scale, and has advantage of producing larger amount of
recombinant OPH than E. coli. The plasmid encoding
recombinant OPH constructed in E. coli could be trans-
Downloaded by [Umeå University Library] at 05:22 06 February 2016
ferred to S. fuliginis using the DNA region with repli-
cation function originated from S. amiense.16) This
replication region is expected to be used also for other
recombinant plasmids encoding useful genes such as
those of degradation enzymes of xenobiotics in order
to express them in the species of Sphingomonadaceae.
Author contribution
KN, TO, YS, and KK designed the experiments;
KN, TO, SI, NI performed the experiments and ana-
lyzed the data; KN, YS, and KK wrote the manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was undertaken in part under a research program
sponsored by the Special Coordination Funds for Promoting Science
and Technology, and a research program sponsored by the JSPS
Grant-in Aid for Scientific Research (C), supported by the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. grant
number [23590163].
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