Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

Article abstract of DOI:10.1016/j.molcatb.2013.05.003

5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.

Full text of DOI:10.1016/j.molcatb.2013.05.003

Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with
NADH regeneration
a
a
b
b
Toshiki Furuya , Takaaki Kanno , Hiroaki Yamamoto , Norihiro Kimoto ,
b
a,∗
Akinobu Matsuyama , Kuniki Kino
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
Green Product Development Center, Daicel Corporation, Niigata 944-8550, Japan
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane
derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone
using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells
that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that
co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate
NADH using glucose. Two types of cells – wet cells that did not receive any treatment after washing with
glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing – were pre-
pared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing
only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the pres-
ence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance
the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis
would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried
cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently
catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh com-
pensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged
by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with
repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system
and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained
high-yield production; the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM
Received 24 December 2012
Received in revised form 5 May 2013
Accepted 7 May 2013
Available online 27 May 2013
Keywords:
Glucose dehydrogenase
Monooxygenase
NADH regeneration
P450cam
Whole-cell catalyst
(3.5 g/l) of 5-hydroxy-2-adamantanone, respectively.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
often accompanied by contamination of several other oxygenated
adamantanes, which leads to low yields of product.
Adamantane derivatives are important industrial chemicals that
find wide use in production of pharmaceuticals and polymers
1–3]. 5-Hydroxy-2-adamantanone (5-hydroxyadamantan-2-one)
Cytochrome P450 monooxygenases (P450s) are able to catalyze
oxidation using molecular oxygen as an innocuous oxidant under
mild reaction conditions. A heme moiety in the catalytic center
of P450 activates molecular oxygen using electrons, which are
transferred from NAD(P)H by reductase components. The resulting
active oxidant, so-called Compound I, oxidizes substrates. P450s
generally introduce one oxygen atom regio- and stereo-selectively
into organic compounds. Hence, they are of considerable interest
as oxidation biocatalysts for the synthesis of industrial chemicals
and pharmaceuticals [8–13]. As mentioned above, P450s require
NAD(P)H for their catalytic activity. Since NAD(P)H is an expensive
reagent, NAD(P)H regeneration systems are essential for cost-
effective biocatalytic oxidation processes [14,15].
[
is a versatile starting material for the synthesis of various 2,5-
or 1,4-disubstituted adamantanes [4]. For example, E-2-amino-5-
hydroxyadamantane, an intermediate for biologically active com-
pounds, can be synthesized from 5-hydroxy-2-adamantanone [5].
Chemical methods for synthesizing 5-hydroxy-2-adamantanone
include disproportionation of 2-hydroxyadamantane and oxida-
tion of 2-adamantanone [4,6,7]. These chemical methods require
harmful oxidants such as 60–75% sulfuric acid, 100% nitric acid, and
chromic oxide. Furthermore, the procedures are elaborate and are
+
NAD(P) -dependent dehydrogenases can function as a system
for the regeneration of NAD(P)H using cheap hydrocarbons as
an energy source [15–17]. These dehydrogenases catalyze the
∗ Corresponding author. Tel.: +81 3 5286 3211; fax: +81 3 3232 3889.
E-mail address: kkino@waseda.jp (K. Kino).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.05.003

1
12
T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
O
Glucose
O
2
-Adamantanone
+
NAD
Glucose
O
2
Reduced
P450cam
Oxidized
Oxidized
Pdx
Reduced
Reduced
PdR
Oxidized
Gdh
+
NADH+H
Gluconate
H
2
O
+
+
NADH+H NAD
O
5
2
-Hydroxy-
-adamantanone
Glycolysis
and TCA cycle
Glucose
HO
Escherichia coli cell
O
Glucose
HO
Fig. 1. Scheme of whole-cell catalyst expressing the P450cam multicomponent system coupled with NADH regeneration system for the regioselective oxidation of 2-
adamantanone. Pdx, putidaredoxin; PdR, putidaredoxin reductase; Gdh, glucose dehydrogenase; TCA, tricarboxylic acid.
elimination of two hydrogen atoms from organic molecules such
as glucose, glycerol, and alcohols, and consequently generate
NAD(P)H from NAD(P) . Several studies have attempted to con-
constructed to regenerate NADH using glucose (Fig. 1). Further-
more, two types of whole cells – wet cells that did not receive
any treatment after washing with glycerol-containing buffer, and
freeze-dried cells that were lyophilized after the washing – were
prepared. We examined the effects of Gdh co-expression on these
P450cam whole-cell catalysts and investigated the application
of these catalysts to a flask-scale production of 5-hydroxy-2-
adamantanone.
+
struct supplying system of NAD(P)H using these dehydrogenases
in pure-enzyme or whole-cell P450 catalyses [15,18–23]. However,
the effects of dehydrogenase co-expression on P450 whole-cell cat-
alysts varied significantly among the reports [18–23].
In this study, we report a synthetic approach to produce 5-
hydroxy-2-adamantanone using P450cam coupled with NADH
regeneration as an oxidation biocatalyst. P450cam (CYP101A1) is a
camphor monooxygenase [24] that has been shown to exhibit oxi-
dation activity towards a variety of compounds including aliphatics
and aromatics [25–27]. It was also confirmed that P450cam has
catalytic activity in the oxidation of 2-adamantanone to 5-hydroxy-
2. Materials and methods
2.1. Bacterial strains, plasmids, cultivation media, and chemicals
The bacterial strains and plasmids that were used or con-
2
-adamantanone using purified enzyme in biochemical studies
structed in this study are listed in Table 1. The bacteria were grown
in Luria–Bertani (LB) medium, which contained (per liter) Bacto
tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g) (pH 7.0). 2-
Adamantanone and 5-hydroxy-2-adamantanone were purchased
from Tokyo Kasei (Tokyo, Japan). All other chemicals were of ana-
lytical grade.
[
28]. In the application of P450s to biocatalysis, whole-cell cata-
lysts have several advantages compared with purified enzyme. For
example, enzymes in cells are protected from the external envi-
ronment and often more stable than purified enzymes [29–31].
This stability provides longer lifetime for whole-cell catalysts. In
addition, when enzymes are multicomponent, it is easier and less
expensive to reconstitute their catalytic activity in vivo than in vitro
[
32]. Here, we constructed genetically engineered Escherichia coli
2.2. Construction of P450cam, pdx, pdR, and gdh expression
plasmids
cells expressing P450cam multicomponent system consisting of
P450cam and its redox partners, putidaredoxin (Pdx) and puti-
daredoxin reductase (PdR) (Fig. 1). E. coli cells co-expressing this
P450cam system with a glucose dehydrogenase (Gdh) was also
The plasmids used for expression of the P450cam and pdx
genes in E. coli cells were constructed using the pETDuet-1 vector
Table 1
Bacterial strains and plasmids used in this study.
Strain or plasmid
Characteristics
Reference or source
Strains
E. coli JM109
E. coli BL21 (DE3)
Plasmids
pETDuet-1
pACYCDuet-1
pETDpdx
Host used for cloning
Host used for expression
Takara Bio
Novagen
Vector used for cloning, Apr
Vector used for cloning, Cm
Novagen
Novagen
r
pETDuet-1 containing pdx in MCS-2 under the control
of the T7 promoter
pETDuet-1 containing P450cam in MCS-1 and pdx in
MCS-2 under the control of the T7 promoters
pACYCDuet-1 containing pdR in MCS-1 under the
control of the T7 promoter
This study
This study
This study
This study
pETDP450cam-pdx
pACYCDpdR
pACYCDpdR-gdh
pACYCDuet-1 containing pdR in MCS-1 and gdh in
MCS-2 under the control of the T7 promoters

T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
113
Table 2
◦
and pACYCDpdR-gdh were cultivated at 30 C in LB medium
supplemented with ampicillin (100 ␮g/ml) and chlorampheni-
col (100 ␮g/ml). After cultivation for 6 h (OD600 = 0.8–1.0),
isopropyl-␤-d-thiogalactopyranoside (1 mM), 5-aminolevulinic
acid (0.5 mM), and FeSO4 (0.5 mM) were added to the medium, and
Oligonucleotide primers used in this study.
ꢀ
ꢀ a
Primer
Sequence (5 –3 )
Restriction
site
pdx-F
pdx-R
P450cam-F
P450cam-R
pdR-F
pdR-R
gdh-F
gdh-R
TTC CAT ATG TCT AAA GTA GTG TAT GTG TCA
GA AGA TCT TTA CCA TTG CCT ATC GGG AAC
TTC TCA TGA CGA CTG AAA CCA TAC AAA GCA
CCG GAA TTC TTA TAC CGC TTT GGT AGT CGC
TTC TCA TGA ACG CAA ACG ACA ACG TGG TCA
CGC GGA TCC TCA GGC ACT ACT CAG TTC AGC
TTC CAT ATG TAT CCG GAT TTA AAA GGA AAA
GA AGA TCT TTA ACC GCG GCC TGC CTG GAA
NdeI
BglII
◦
cultivation was continued for an additional 15 h at 25 C. Cells were
BspHI
EcoRI
BspHI
BamHI
NdeI
harvested by centrifugation and washed with potassium phosphate
buffer (200 mM, pH 7.5) containing glycerol (10%, v/v). These cells
were used as wet cells. After treatment with lyophilizer (FDU-1000,
EYELA, Tokyo, Japan), the resulting cells were used as freeze-dried
cells.
BglII
a
Restriction sites are underlined and identified in the column on the right. Initi-
ation and termination codons are indicated in bold.
2.4. Protein and P450 analyses
(
Table 1). Two oligonucleotide primers, pdx-F and pdx-R (Table 2),
When cell-free extracts were prepared, cells were suspended in
potassium phosphate buffer (50 mM, pH 7.5) containing glycerol
(10%, v/v) and were disrupted by sonication. After centrifugation
at 15,000 × g for 30 min at 4 C, the resulting supernatant was used
were designed to amplify the pdx gene (GenBank accession num-
ber, P00259). The region between the two oligonucleotide primers
was amplified from the pET21a vector carrying the pdx gene [33] by
PCR. PCR was performed with KOD Plus polymerase (Toyobo, Osaka,
Japan) under the buffer conditions recommended by the manu-
facturer. The PCR mixture was heated at 94 C for 2 min and then
subjected to 30 cycles of amplification (94 C for 15 s; 55 C for 30 s;
and 68 C for 30 s). This amplified DNA fragment was digested with
NdeI and BglII, and then inserted into the multi-cloning site (MCS)-2
of the pETDuet-1 vector that was digested with the same restric-
tion enzymes. The resulting plasmid, pETDpdx, was amplified in
E. coli JM109 cells. Next, two oligonucleotide primers, P450cam-F
and P450cam-R (Table 2), were designed to amplify the P450cam
gene (GenBank accession number, P00183). The region between
the two oligonucleotide primers was amplified from genomic DNA
of Pseudomonas putida JCM 6157 (ATCC 17453) by PCR. The PCR
◦
as the cell-free extract. The cell-free extracts were used to ana-
lyze protein levels and P450 expression. Protein concentration was
measured using a Coomassie protein assay kit (Pierce, Rockford, IL,
USA) with a bovine serum albumin standard [34]. The expression
levels of P450cam, Pdx, PdR, and Gdh were examined by sodium
dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE)
analysis. Active P450cam was quantified based on the reduced CO
difference spectrum characteristic of thiolate-heme enzymes using
◦
◦
◦
◦
−
1
−1
an extinction coefficient of 91 mM cm at 450 nm [35]. Before
analysis of these spectra, the protein concentration of the cell-free
extracts was adjusted to 2 g/l.
2.5. Reactions using whole cells
◦
mixture was subjected to 30 cycles of amplification (94 C for 15 s;
◦
◦
6
5 C for 30 s; and 68 C for 90 s). This amplified DNA fragment
was digested with BspHI and EcoRI, and then inserted into MCS-
of the pETDpdx plasmid that was digested with NcoI and EcoRI.
The reaction mixture (50 ml) contained cells of the transformed
E. coli strain (15 g dry cell weight (DCW)/l), 2-adamantanone
(20 mM), dimethylsulfoxide (DMSO) (10%, v/v), and potassium
phosphate buffer (200 mM, pH 7.5) containing glycerol (10%, v/v).
The reaction mixture was supplemented with glucose (50 mM)
when required. Fifteen grams of DCW corresponded to 50 g of wet
1
The resulting plasmid, pETDP450cam-pdx, was amplified in E. coli
JM109 cells.
The plasmids used for expression of the pdR and gdh genes
in E. coli cells were constructed using the pACYCDuet-1 vector
◦
cell weight. The reactions were performed at 30 C with rotary
(Table 1). Two oligonucleotide primers, pdR-F and pdR-R (Table 2),
shaking at a speed of 120 rpm.
were designed to amplify the pdR gene (GenBank accession num-
ber, P16640). The region between the two oligonucleotide primers
was amplified from the pET21a vector carrying the pdR gene [33]
by PCR. The PCR mixture was subjected to 30 cycles of amplification
2.6. Production of 5-hydroxy-2-adamantanone with repeated
additions of the substrate
◦
◦
◦
(
94 C for 15 s; 60 C for 30 s; and 68 C for 90 s). This amplified DNA
The reaction was performed in a 500-ml flask that con-
tained cells of the transformed E. coli strain (15 g DCW/l),
2-adamantanone (20 mM), glucose (50 mM), and potassium phos-
phate buffer (200 mM, pH 7.5) containing glycerol (10%, v/v) in a
volume of 50 ml. After 4-h incubation, 2-adamantanone (20 mM)
and glucose (50 mM) were again added to the reaction mixture.
fragment was digested with BspHI and BamHI, and then inserted
into MCS-1 of the pACYCDuet-1 vector that was digested with NcoI
and BamHI. The resulting plasmid, pACYCDpdR, was amplified in E.
coli JM109 cells. Next, two oligonucleotide primers, gdh-F and gdh-
R (Table 2), were designed to amplify the gdh gene (GenBank acces-
sion number, NP 388275). The region between the two oligonu-
cleotide primers was amplified from genomic DNA of Bacillus
subtilis strain 168 (ATCC 23857) by PCR. The PCR mixture was sub-
◦
The reactions were carried out at 30 C with rotary shaking at a
speed of 120 rpm.
◦
◦
jected to 30 cycles of amplification (94 C for 15 s; 60 C for 30 s; and
6
2.7. Product analysis and glucose measurement
◦
8 C for 60 s). This amplified DNA fragment was digested with NdeI
and BglII, and then inserted into MCS-2 of the pACYCDpdR plasmid
that was digested with the same restriction enzymes. The resulting
plasmid, pACYCDpdR-gdh, was amplified in E. coli JM109 cells.
After the plasmid constructs were confirmed by sequencing,
these plasmids were introduced into E. coli BL21 (DE3) cells by
electroporation.
Gas chromatography (GC) analysis was performed using a GC-
2010 system (Shimadzu, Kyoto, Japan) equipped with a flame
ionization detector and a 30-m type DB-5 column (J&W Scientific,
Folsom, CA, USA). A portion of the reaction mixture was acidi-
fied by the addition of HCl (pH 2–3) and was extracted with ethyl
acetate. The resulting extract was injected into the GC system.
The carrier gas was helium and the flow rate was 1.0 ml/min. The
◦
2.3. Preparation of whole cells
injection temperature was maintained at 250 C. The oven tem-
◦
perature was programmed as follows: start temperature of 120 C,
◦
◦
The transformed E. coli BL21 (DE3) cells carrying pETDP450cam-
pdx and pACYCDpdR, and the cells carrying pETDP450cam-pdx
increased to 165 C at a rate of 15 C/min, further increased to
◦ ◦
180 C at a rate of 5 C/min, and held for 4 min at finish temperature

1
14
T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
Fig. 2. Expression analysis of P450cam, Pdx, PdR, and Gdh in E. coli. (A) SDS-PAGE analysis. Samples (5 ␮g of cell lysate) from the soluble fractions of the transformed E. coli
cells were loaded onto a polyacrylamide gel (20%). Lane 1, cells carrying pETDuet-1; lane 2, pETDpdx; lane 3, pETDP450cam-pdx; lane 4, pACYCDuet-1; lane 5, pACYCDpdR;
lane 6, pACYCDpdR-gdh; lane 7, pETDP450cam-pdx and pACYCDpdR; lane 8, pETDP450cam-pdx and pACYCDpdR-gdh. The molecular weights corresponding to P450cam,
Pdx, PdR, and Gdh are indicated by arrows. (B) CO-reduced difference spectra. Samples (2 mg protein/ml) from the soluble fractions of the transformed E. coli cells were
analyzed. Solid line, cells carrying pETDP450cam-pdx and pACYCDpdR; dotted line, cells carrying pETDP450cam-pdx and pACYCDpdR-gdh.
◦
of 180 C. The amounts of 2-adamantanone and 5-hydroxy-2-
adamantanone were calculated from standard calibration curves
that were made using these compounds purchased from Tokyo
Kasei. Gas chromatography–mass spectrometry (GC–MS) analysis
was performed using a JMS-GCmateII system (JEOL, Tokyo, Japan)
with a DB-5 column. Ions were generated using an electron ioniza-
tion source, and the mass detector was tuned using 2-admantanone
to optimize the instrumental parameters. Reaction products were
identified based on GC–MS spectra.
and pdR genes produced 0.56 ␮mol P450cam per liter of culture
(0.093 ␮mol P450cam per g of wet cells), and E. coli cells carrying
the P450cam, pdx, pdR, and gdh genes produced 0.57 ␮mol P450cam
per liter of culture (0.097 ␮mol P450cam per g of wet cells).
3.2. Biocatalytic oxidation of 2-adamantanone using wet E. coli
cells expressing P450cam
We explored the catalytic potential of P450cam for the oxi-
dation of 2-adamantanone. Wet cells that did not receive any
treatment after washing with glycerol-containing buffer were
first used for whole-cell reactions (Fig. 3). E. coli cells that car-
ried the P450cam, pdx, and pdR genes, but not the gdh gene,
were reacted with 20 mM 2-adamantanone. The reaction mix-
ture was supplemented with 50 mM glucose. GC analysis of the
reactions with 2-adamantanone showed a major peak (retention
time, 6.5 min) in addition to the substrate peak (4.6 min) (see
Fig. 4A). The retention time and the GC–MS spectrum of this
product coincided with those of an authentic sample of 5-hydroxy-
2-adamantanone. Based on these observations, this product was
identified as 5-hydroxy-2-adamantanone, as reported previously
using purified enzyme [28]. The transformed E. coli cells oxidized
2-adamantanone regioselectively at the C-5 position and produced
5.4 mM 5-hydroxy-2-adamantanone within 8 h in the absence of
glucose (Fig. 3A). Furthermore, addition of glucose to the reaction
mixture strongly enhanced the amount of product; the whole-cell
catalyst produced 19 mM 5-hydroxy-2-adamantanone within 8 h
in the presence of glucose (Fig. 3B). These results suggest that NADH
Glucose concentration was measured using a Glucose CII-test
kit (Wako, Osaka, Japan) according to the instruction manual.
3
. Results and discussion
3.1. Preparation of P450cam whole-cell catalysts
We constructed E. coli cells that expressed P450cam and its
redox partners, Pdx and PdR, and cells that co-expressed this
P450cam multicomponent system with Gdh. The P450cam and pdx
genes were cloned into the pETDuet-1 vector, and the pdR and gdh
genes were cloned into the pACYCDuet-1 vector. The expression of
these genes in E. coli BL21 (DE3) cells was induced by isopropyl-
␤
-d-thiogalactopyranoside under the control of their respective
T7 promoters. SDS-PAGE analysis showed the major bands corre-
sponding to P450cam, Pdx, PdR, and Gdh in the soluble fractions
of the transformed E. coli cells (Fig. 2A). The expression levels of
P450cam were also determined based on the reduced CO differ-
ence spectrum (Fig. 2B); E. coli cells carrying the P450cam, pdx,

T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
115
(
A) 25
(B) 25
50
40
30
20
10
0
2
1
1
0
5
0
20
15
10
5
5
0
0
0
4
8
12
Time (h)
16
20
24
0
4
8
12
Time (h)
16
20
24
(
C) 25
(D) 25
50
40
30
20
10
0
2
1
1
0
5
0
20
15
10
5
5
0
0
0
4
8
12
Time (h)
16
20
24
0
4
8
12
Time (h)
16
20
24
Fig. 3. Biocatalytic oxidation of 2-adamantanone using wet whole-cell catalysts. E. coli cells expressing only the P450cam multicomponent system (A and B) and E. coli cells
co-expressing this P450cam system with Gdh (C and D) were reacted with 2-adamantanone in the absence (A and C) or the presence (B and D) of glucose. The time courses of
2
-adamantanone consumption (open circles) and of 5-hydroxy-2-adamantanone production (closed circles) are shown. Plots indicate the average values of determinations
performed in double, and error bars indicate standard deviation from the mean. In (B) and (D), glucose consumption (triangles) is also shown.
was effectivelyregeneratedthrough endogenousglucose metabolic
pathways such as glycolysis and the tricarboxylic acid cycle in the
E. coli host.
results suggest that 5-hydroxy-2-adamantanone was oxidized
to dihydroxy-2-adamantanone by P450cam, although elucidation
of this reaction needs detailed characterization using purified
enzyme.
This product, however, gradually decreased after 8 h in the pres-
ence of glucose (Fig. 3B). By GC analysis, a small peak (retention
time, 7.0 min) and a trace of peak (8.6 min) were newly detected
after 8 h (Fig. 4A). We confirmed that these two peaks were also
detected after the reaction using 5-hydroxy-2-adamantanone as a
substrate (data not shown). By GC–MS analysis, the m/z value of
parent ion of the product at 7.0 min was found to be 168 (Fig. 4B).
This value corresponds to the compound formed from 5-hydroxy-
Whole-cell reactions were also performed using wet E. coli
cells co-expressing the P450cam multicomponent system with
Gdh. The transformed E. coli cells produced 17 mM 5-hydroxy-
2-adamantanone within 8 h in the presence of glucose (Fig. 3D).
E. coli cells co-expressing Gdh consumed glucose more rapidly
than cells without the gdh gene (Fig. 3B and D). The rate of 5-
hydroxy-2-adamantanone production, however, was almost the
same between these two transformed E. coli strains (Fig. 3B and D).
These results indicate that enough amounts of NADH for P450cam
catalysis would be supplied by endogenous glucose metabolism in
the E. coli host.
2
-adamantanone (molecular weight, 166) through two-hydrogen
addition. Furthermore, the GC–MS spectrum of this product coin-
cided with that of 1,4-adamantanediol that had been previously
determined [36]. These observations strongly suggest that the
product at 7.0 min would be 1,4-adamantandiol. We also confirmed
that E. coli cells without the P450cam gene were able to con-
vert 5-hydroxy-2-adamantanone to 1,4-adamantanediol (data not
shown). These results indicate that 5-hydroxy-2-adamantanone
was reduced to 1,4-adamantanediol (the compound correspond-
ing to 2,5-adamantanediol) by an endogenous enzyme in the E. coli
host. On the other hand, the parent ion of the product at 8.6 min was
m/z 182 (Fig. 4C). This value corresponds to the compound formed
from 5-hydroxy-2-adamantanone through one-oxygen addition.
Furthermore, the fragment ions at m/z 95 and m/z 111, which are
typical for dihydroxylated adamantanes [36], were also detected.
Based on these observations, the product at 8.6 min was tenta-
tively identified as dihydroxy-2-adamantanone. This product was
not detected when E. coli cells without the P450cam gene were
reacted with 5-hydroxy-2-adamantanone (data not shown). These
3.3. Biocatalytic oxidation of 2-adamantanone using freeze-dried
cells expressing P450cam
Freeze-dried cells that were lyophilized after washing with
glycerol-containing buffer were next used for whole-cell reactions
(Fig. 5). E. coli cells carrying the P450cam, pdx, and pdR genes, but
not the gdh gene, were reacted with 20 mM 2-adamantanone. The
reaction mixture was supplemented with 50 mM glucose. However,
the transformed E. coli cells produced hardly any 5-hydroxy-2-
adamantanone even in the presence of glucose (Fig. 5B).
Whole-cell reactions were also performed using freeze-dried E.
coli cells co-expressing the P450cam multicomponent system with
Gdh. The transformed E. coli cells produced hardly any 5-hydroxy-
2-adamantanone in the absence of glucose (Fig. 5C). In contrast,

1
16
T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
Fig. 4. GC–MS analysis of by-products generated by the P450cam whole-cell reaction. Wet E. coli cells that carried the P450cam, pdx, and pdR genes were reacted with 20 mM
-adamantanone in the presence of glucose for 24 h. (A) GC chromatogram. The peak at 6.5 min was found to correspond to 5-hydroxy-2-adamantanone. (B) GC–MS spectrum
for the peak at 7.0 min in chromatogram (A). (C) GC–MS spectrum for the peak at 8.6 min in chromatogram (A).
2
these cells rapidly oxidized the substrate in the presence of glu-
cose; 16 mM 5-hydroxy-2-adamantanone was produced within 8 h,
and this production was accompanied by rapid consumption of
glucose (Fig. 5D). These results suggest that endogenous glucose
metabolism in the E. coli host would be damaged by the lyophiliza-
tion process, and thus the freeze-dried cells required co-expression
of Gdh for the oxidation activity.
Interestingly, 5-hydroxy-2-adamantanone was not further
transformed by the freeze-dried cells after 8 h (Fig. 5D). This result
was different from that observed for the reaction using wet cells
again added. Although 2-adamantanone was added as a DMSO
solution in the experiments presented above, the solid substrate
was directly added to the reaction mixture to avoid the toxic-
ity of the organic solvent DMSO in this process. As shown in
Fig. 6A, the whole-cell catalyst completely transformed 20 mM 2-
adamantanone into 5-hydroxy-2-adamantanone within 4 h. The
initial rate of 5-hydroxy-2-adamantanone production, estimated
−
1
−1
for the first 1 h of the reaction, was 52 mol (mol P450) min
−
1
−1
(16 ␮mol g-DCW min ). This rate was approximately 1.3 times
higher compared with that of 5-hydroxy-2-adamantanone pro-
duction in the presence of DMSO (Fig. 3B). Furthermore, after
the second addition of substrate, the production of 5-hydroxy-2-
adamantanone continued to increase. Within 24 h of reaction, the
amount of this product reached 36 mM (5.9 g/l), with a molar con-
version yield of 90%.
(Fig. 3B and D), suggesting that an E. coli enzyme that catalyzed the
reduction of 5-hydroxy-2-adamantanone to 1,4-adamantanediol
might be deactivated by the lyophilization process. This method
might be widely applicable to whole-cell reactions accompanied
by undesired side reactions derived from host cells.
Using a similar technique, we also attempted to produce
5-hydroxy-2-adamantanone with freeze-dried E. coli cells co-
expressing the P450cam multicomponent system with Gdh
3
.4. Production of 5-hydroxy-2-adamantanone with repeated
additions of the substrate
(Fig. 6B). This whole-cell catalyst produced 16 mM 5-hydroxy-
The results presented above demonstrate that E. coli cells car-
rying the P450cam, pdx, and pdR genes might be an efficient
biocatalyst for the oxidation of 2-adamantanone. We thus first
attempted to produce 5-hydroxy-2-adamantanone using these
wet cells in a 500-ml flask containing 50 ml of the reaction mix-
ture. The substrate 2-adamantanone (20 mM) and glucose (50 mM)
were initially added to the reaction mixture and, after 4-h incu-
bation, the same amounts of 2-adamantanone and glucose were
2-adamantanone within 4 h. The initial rate of 5-hydroxy-2-
adamantanone production was estimated to be 49 mol (mol
P450) min (16 ␮mol g-DCW min ). Furthermore, after the
second addition of substrate, the amount of this product reached
21 mM (3.5 g/l).
The production rate decreased after 6 h in the wet-cell reaction
and after 5 h in the freeze-dried-cell reaction (Fig. 6). When the
pH of the reaction mixture dropped below 6.5, the production rate
−
1
−1
−1
−1

T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
117
(
A) 25
(B) 25
50
40
30
20
10
0
2
1
1
0
5
0
20
15
10
5
5
0
0
0
4
8
12
Time (h)
16
20
24
0
4
8
12
Time (h)
16
20
24
(
C) 25
(D) 25
50
40
30
20
10
0
2
1
1
0
5
0
20
15
10
5
5
0
0
0
4
8
12
Time (h)
16
20
24
0
4
8
12
Time (h)
16
20
24
Fig. 5. Biocatalytic oxidation of 2-adamantanone using freeze-dried whole-cell catalysts. E. coli cells that expressing only the P450cam multicomponent system (A and B)
and E. coli cells co-expressing this P450cam system with Gdh (C and D) were reacted with 2-adamantanone in the absence (A and C) or the presence (B and D) of glucose. The
time courses of 2-adamantanone consumption (open circles) and of 5-hydroxy-2-adamantanone production (closed circles) are shown. Plots indicate the average values of
determinations performed in double, and error bars indicate standard deviation from the mean. In (B) and (D), glucose consumption (triangles) is also shown.
(
A) 60
8
7
6
5
4
(B) 60
8
7
6
5
4
5
4
3
2
1
0
0
0
0
0
50
40
30
20
10
0
0
0
4
8
12
Time (h)
16
20
24
0
4
8
12
Time (h)
16
20
24
Fig. 6. Production of 5-hydroxy-2-adamantanone with repeated additions of the substrate. Wet E. coli cells expressing only the P450cam multicomponent system (A) and
freeze-dried E. coli cells co-expressing this P450cam system with Gdh (B) were reacted with the substrate 2-adamantanone in the presence of glucose. The time courses of
5
-hydroxy-2-adamantanone production (closed circles), glucose consumption (triangles), and pH (squares) are shown. 2-Adamantanone (20 mM) and glucose (50 mM) were
initially added to the reaction mixture and, after 4-h incubation, the same amounts of 2-adamantanone and glucose were again added.
decreased in both wet cells and freeze-dried cells (Fig. 6). It was
reported that P450cam exhibits low oxidation activity under acidic
conditions lower than pH 6.5 [37]. These results suggest that the
decrease of production rate might be caused by the decrease of
pH. However, although the pH decreased more rapidly in wet cells
than in freeze-dried cells, the production after the second addition
of substrate was higher in wet cells (Fig. 6), suggesting that other
factors also affected the production. Detailed characterization will
be necessary for further enhancement of productivity.
4. Conclusions
We succeeded in producing 36 mM (5.9 g/l) 5-hydroxy-2-
adamantanone using wet cells expressing only the P450cam
multicomponent system (Fig. 6A) and 21 mM (3.5 g/l) of the product
using freeze-dried cells co-expressing this P450cam system with
Gdh (Fig. 6B). The oxidation activities of the whole cells towards
2-adamantanone were almost the same as that of the purified
−
1
−1
P450cam system previously reported (52 mol (mol P450) min )

1
18
T. Furuya et al. / Journal of Molecular Catalysis B: Enzymatic 94 (2013) 111–118
[
28], whereas our experimental setup using whole cells achieved
[14] E. O’Reilly, V. Köhler, S.L. Flitsch, N.J. Turner, Chem. Commun. (Camb.) 47 (2011)
490–2501.
2
gram-per-liter-scale production of 5-hydroxy-2-adamantanone.
Through the experiments, we found that wet cells were able to
effectively oxidize 2-adamantanone without co-expression of Gdh,
whereas freeze-dried cells required co-expression of Gdh for the
oxidation activity (Figs. 3 and 5). In other words, these results
demonstrate that the condition of the host cells markedly affects
the NADH regeneration system used in whole-cell oxidation catal-
ysis (Fig. 1). Wet cells, which attained higher-level production
than freeze-dried cells, are more desirable for use immediately
after the cell preparation. On the other hand, freeze-dried cells
repressed undesired side reactions derived from host cells, and
may be advantageous in the preservation of the whole-cell cata-
lyst. These efficient whole-cell catalysts might pave the way for
industrial biocatalytic production of 5-hydroxy-2-adamantanone.
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