Molecular cloning and functional characterization of a two highly stereoselective borneol dehydrogenases from Salvia officinalis L

Article abstract of DOI:10.1016/j.phytochem.2019.112227

Enzymes for selective terpene functionalization are of particular importance for industrial applications. Pure enantiomers of borneol and isoborneol are fragrant constituents of several essential oils and find frequent application in cosmetics and therapy. Racemic borneol can be easily obtained from racemic camphor, which in turn is readily available from industrial side-streams. Enantioselective biocatalysts for the selective conversion of borneol and isoborneol stereoisomers would be therefore highly desirable for their catalytic separation under mild reaction conditions. Although several borneol dehydrogenases from plants and bacteria have been reported, none show sufficient stereoselectivity. Despite Croteau et al. describing sage leaves to specifically oxidize one borneol enantiomer in the late 70s, no specific enzymes have been characterized. We expected that one or several alcohol dehydrogenases encoded in the recently elucidated genome of Salvia officinalis L. would, therefore, be stereoselective. This study thus reports the recombinant expression in E. coli and characterization of two enantiospecific enzymes from the Salvia officinalis L. genome, SoBDH1 and SoBDH2, and their comparison to other known ADHs. Both enzymes produce preferentially (+)-camphor from racemic borneol, but (?)-camphor from racemic isoborneol.

Full text of DOI:10.1016/j.phytochem.2019.112227

Phytochemistry172(2020)112227
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Molecular cloning and functional characterization of a two highly
stereoselective borneol dehydrogenases from Salvia officinalis L
Ivana Drienovskáa, Dajana Kolanovića, Andrea Chániquea,b, Volker Sieberc,d, Michael Hoferc,
Robert Kourista,∗
a Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria
b Department of Chemical and Bioprocesses Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Macul, 7820436,
Santiago, Chile
c Fraunhofer Institute for Interfacial Engineering and Biotechnology Bio, Electro and Chemocatalysis BioCat, Straubing Branch, Schulgasse 11a, 94315, Straubing, Germany
d Technische Universität München TUM, Campus Straubing für Biotechnologie und NachhaltigkeitSchulgasse 16, 94315 Straubing, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Salvia officinalis L.
Lamiaceae
Selective oxidation
Monoterpenes
Borneol dehydrogenases
Borneol
Enzymes for selective terpene functionalization are of particular importance for industrial applications. Pure
enantiomers of borneol and isoborneol are fragrant constituents of several essential oils and find frequent ap-
plication in cosmetics and therapy. Racemic borneol can be easily obtained from racemic camphor, which in turn
is readily available from industrial side-streams. Enantioselective biocatalysts for the selective conversion of
borneol and isoborneol stereoisomers would be therefore highly desirable for their catalytic separation under
mild reaction conditions. Although several borneol dehydrogenases from plants and bacteria have been reported,
none show sufficient stereoselectivity. Despite Croteau et al. describing sage leaves to specifically oxidize one
borneol enantiomer in the late 70s, no specific enzymes have been characterized. We expected that one or
several alcohol dehydrogenases encoded in the recently elucidated genome of Salvia officinalis L. would,
therefore, be stereoselective. This study thus reports the recombinant expression in E. coli and characterization of
two enantiospecific enzymes from the Salvia officinalis L. genome, SoBDH1 and SoBDH2, and their comparison to
other known ADHs. Both enzymes produce preferentially (+)-camphor from racemic borneol, but (−)-camphor
from racemic isoborneol.
Camphor
1. Introduction
of synthetic routes for the synthesis of optically pure borneol a desirable
goal (Niu et al., 2018; Zhang et al., 2017). Biocatalysis offers environ-
Monoterpenes constitute a large and diverse group of hydrocarbons
found in nature which boast antibacterial, antifungal and anticancer
properties making them of high interest for chemical and pharmaceu-
tical industries (Holstein and Hohl, 2004). Enzymes for terpene meta-
bolism can be classified into two main groups: enzymes for the selective
formation of the skeleton (Dickschat, 2016) and enzymes for selective
terpene functionalization (Brück et al., 2014; Wriessnegger et al.,
2014). The latter group is of particular importance since it allows the
functionalization of terpenes from industrial side-streams (Hofer et al.,
2013; Oberleitner et al., 2017). Borneol-1 and isoborneol-2 are fragrant
constituents of several essential oils with a bitter taste. With various
bioactivities, both compounds have been used as Borneolum ((+)-1)
and Borneolum syntheticum (a mixture of racemic 1 and 2) in tradi-
tional Chinese medicine (Cheng et al., 2013). A large number of ap-
plications in cosmetics, fragrance and medicine make the development
mental benefits in many cases; particularly, the outstanding selectivity
of Nature's catalysts has led to numerous applications in the synthesis of
enantiomerically enriched fine chemicals. Racemic borneol can be ea-
sily produced from racemic camphor, which in turn is a cheap waste
material from pine tree processing; therefore, enzymes for its selective
functionalization are of particular interest. Several alcohol dehy-
drogenases accepting borneol were isolated from oil-bearing plants
such as Lavandula intermedia and Artemisia annua (Polichuk et al., 2010;
Sarker et al., 2012; Tian et al., 2015). In addition, a bacterial enzyme
from Pseudomonas sp. TCU-HL1 has recently been reported to perform
the oxidation of borneol (Tsang et al., 2016). Enzymatic oxidation of
racemic borneol would be a simple approach for the synthesis of en-
antiomerically pure borneol and camphor. Yet, all recombinantly pro-
duced enzymes so far do not show any enantioselectivity. Fig. 1 shows
the role of ketoreductases in the biosynthesis of (+)-camphor (Croteau
∗ Corresponding author.
E-mail address: kourist@tugraz.at (R. Kourist).
https://doi.org/10.1016/j.phytochem.2019.112227
Received 6 July 2019; Received in revised form 26 November 2019; Accepted 17 December 2019
0031-9422/©2020ElsevierLtd.Allrightsreserved.

I. Drienovská, et al.
Phytochemistry172(2020)112227
Fig. 1. Proposed pathway for (+)-camphor biosynthesis. BPC: (+)-bornyl pyrophosphate cyclase, BPPH: (+)-bornyl pyrophosphate hydrolase, BDH: (+)-borneol
dehydrogenase.
and Karp, 1979a, 1979b; Croteau and Felton, 1980). Notably, as a
dedicated monoterpene synthase produces (+)-borneol, enantioselec-
tivity towards borneol is not required for natural biosynthesis.
In 1978, prior to the characterization of recombinant borneol de-
hydrogenases, Croteau and colleagues reported the selective oxidation
of (+)-borneol to (+)-camphor by the leaf homogenate from of Salvia
officinalis, indicating the presence of selective alcohol dehydrogenases.
Partial purification of leaves by gel permeation and affinity chroma-
tography revealed the presence of enzymes with a high selectivity to-
wards (+)-borneol (Croteau et al., 1978). To our knowledge, all
available recombinant ADHs do not show significant selectivity for
camphor or borneol enantiomers; thus, a selective alcohol dehy-
drogenase that can allow for straightforward catalytic separation by
selective conversion of one enantiomer, would be beneficial.
2. Results and discussion
Intrigued by the possibility of a simple separation of borneol iso-
Fig. 2. Phylogenetic analysis of SoBDH1, SoBDH2 and related ADHs. The tree
was constructed using the neighbour-joining algorithm with Clustal O and vi-
sualized with Mega 7. Enzyme abbreviation, NCBI-accession number and spe-
cies are following (enzymes used in this study marked in bold): LiBDH
mers, we reasoned that the genome of sage should contain alcohol
dehydrogenases with putative activity and selectivity towards borneol.
As racemic mixtures of borneol isomers are easily available from
pinene-derived camphor, a selective alcohol-dehydrogenase would
allow access to pure borneol enantiomers and thus provide a simple
route for the synthesis of bio-based optically pure chemicals. Our as-
sumption was that the dehydrogenases in sage could possibly share a
common ancestor with the non-selective borneol-oxidizing enzymes
from Artemisia annua and Lavandula intermedia. Therefore, a similarity
analysis, recombinant expression and characterization of putative de-
hydrogenases in sage would potentially identify the selective enzyme
reported by the Croteau group (Croteau et al., 1978).
A translated protein sequence of borneol dehydrogenase from A.
annua (GenBank accession ADK56099.1) was blasted (PBLAST) against
the 1000 plants database (1 KP), which contains the Salvia officinalis L.
genome (Matasci et al., 2014). Using this approach, we were able to
identify several sequences with high protein sequence identity to
known enzymes. The top hit showed 77% identity and 90% similarity
(amino acids 148–273), however, it only contained the second half of
the protein and was missing the conserved N-terminal region of dehy-
drogenases, therefore it was excluded from further analysis and char-
acterization. The second two top hits were amino acid sequences with
52% and 51% sequence identity, 68% and 67% similarity. Genes en-
coding for these enzymes are here and further referred to as SoBDH1
and SoBDH2. SoBDH1 and SoBDH2 encode polypeptide chains of 259
and 283 amino acid residues, with a predicted molecular mass of
26.4 kDa and 30.1 kDa, respectively. Noteworthy, these two enzymes
share merely 48% sequence identity towards each other.
(LiBDH_0259)
-
borneol dehydrogenase from Levandula intermedia
(AFV30207.1), AaBDH (AaBDH_0294) - borneol dehydrogenase from Artemisia
annua (ANJ65952.1), ADH2 (AaADH_0265) - alcohol dehydrogenase from
Artemisia annua (ADK56099.1), PsBDH (PsBDH_0261) - borneol dehydrogenase
from Pseudomonas sp. TCU-HL1, SoBDH1 (SoBDH1_0259) - borneol dehy-
drogenase 1 from Salvia officinalis L., SoBDH2 (SoBDH2_0283) - borneol de-
hydrogenase 2 from Salvia officinalis L., PySDH_0270 - Secoisolariciresinol de-
hydrogenase from Prunus yedoensis var. nudiflora (PQQ15129.1), AaBDH2_0293
- borneol dehydrogenase from Artemisia annua (PWA65158.1), AaADH2_0265 -
alcohol dehydrogenase from Artemisia annua (PWA54131.1), PpSCDH_0285 -
putative short-chain dehydrogenase/reductase SDR from Pseudomonas putida
(BAN13298.1). EgHYP_0318 - hypothetical protein MIMGU_mgv1a010226mg
from Erythranthe guttata (EYU18089.1), DhSCDH_0290 - short chain alcohol
dehydrogenase from Dorcoceras hygrometricum.
SoBDH2 gives some insight into their evolution. These two enzymes are
related to other dehydrogenases and reductases which act on mono-
terpenoids in a variety of plant families – the observed pattern is con-
sistent with a relatively early divergence. Both sequences share an N-
terminal conserved G-X-X-X-G-X-G (Gly19/27-X-X-X-Gly23/31-X-
Gly25/33) sequence motif for the NAD(H)-binding region (Fig. 3, red
square). Furthermore, a catalytically essential Y-X-X-X-K motif and Ser
residue, which are conserved in many short-chain dehydrogenases, can
also be found (Fig. 3, blue square).
The SoBDH1 and SoBDH2 genes from Salvia officinalis L. genome
were codon-optimized for expression in E. coli and synthetized
(GenScript, USA), these were then subcloned into the expression vectors
pET-41b(+) and pET15b (sequences in Table S1). The resulting plas-
mids pET41b(+):sobdh1 and pET15b:sobdh2 were used to transform E.
coli BL21(DE3). Both genes were found to be well expressed yielding
moderate to high levels of soluble protein (Fig. S1). Afterwards, the
recombinant proteins were extracted and purified by Ni-NTA affinity
column chromatography yielding pure proteins of 2.7 and 24.8 mg/L of
To investigate the evolutionary relationship among SoBDH1,
SoBDH2 and other plant-related alcohol dehydrogenases (ADHs), phy-
logenetic analyses were performed using neighbour-joining method
(program ClustalO) (Sievers et al., 2014). Results (Fig. 2) demonstrate
that SoBDH1 forms cluster with EgHyp from Erythranthe guttata while
SoBDH2 is most closely related to DhSCDh from Dorcoceras hygro-
metricum indicating that both belong to the ‘classical’ subfamily of the
short-chain dehydrogenases. The phylogenetic analysis of SoBDH1 and
2

I. Drienovská, et al.
Phytochemistry172(2020)112227
Fig. 3. Alignment of sequences of Salvia officinalis L. borneol dehydrogenases, SoBDH1 and SoBDH2 and other related plant ADHs. The sequences were aligned using
Clustal O and visualized with Mega 7 (Sievers et al., 2014). NAD(H)-binding region is highlighted in a red square, catalytic residues (S, Y, K) highlighted in a blue
square. Abbreviations of the enzymes is the same as in Fig. 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web
version of this article.)
culture for SoBDH1 and SoBDH2, respectively. The lower expression
yields of SoBDH1 compared to SoBDH2 may be connected to the lower
stability of the first enzyme. We noted that purified SoBDH1 easily
precipitated after long storage periods or when higher concentrations
were reached after the purification. For the direct comparison, we also
expressed the known ADHs from Lavandula intermedia (LiBDH), Arte-
misia annua (AaBDH and ADH2) and from Pseudomonas TCU-HL1
(PsBDH).
The selectivity of SoBDH1 and SoBDH2 during the oxidation of
borneol (Fig. 4A) was initially tested using cell-free extracts of pro-
duced proteins with a mixture of both (+) (1R,2S, 4R)-borneol) and
(−) (1S,2R, 4S-borneol) enantiomers (Table 1, entries 1–7). 1 mM of
each substrate was used with an equimolar concentration of NAD+
cofactor and reactions were run for a maximum of 24 h, with reaction
samples taken at various time points. Reactions were stopped and
evaluated using GC-FID analysis. The oxidation of isoborneol (mixture
of (+) (1S,2S, 4S)-isoborneol and (−) (1R,2R, 4R)-isoborneol) to
camphor was evaluated in the same manner (Fig. 4B, Table 1, entries
7–12). We were pleased to find that both SoBDH1 and SoBDH2 con-
verted (+)-borneol, but not (−)-borneol (Table 1, entries 5 and 6,
Fig. 7A). This confirmed our anticipated idea of selective borneol de-
hydrogenases in the Salvia plant. The other four studied borneol de-
hydrogenases (Table 1, entry 1–4) showed low or no selectivity towards
(+)-borneol, as also previously reported. Furthermore, SoBDH1 and
SoBDH2 showed selectivity towards racemic isoborneol (rac-2), with a
preference for (+)-isoborneol; the selectivity of SoBDH1 was out-
standing (E > 200), while SoBDH2 showed low selectivity (E = 4.6)
(Table 1, entries 11 and 12, Fig. 7B). Surprisingly, the dehydrogenase
from Lavandula intermedia, being unselective towards (+)-borneol,
showed excellent selectivity towards (+)-isoborneol (E
> 200)
(Table 1, entry 7). The fact that dehydrogenases show a preference for
(+)-borneol over (−)-borneol is in line with the putative natural
function in the biosynthesis of (+)-camphor. Therefore, it was some-
what unexpected to observe that most of BDHs converted (+)-iso-
borneol faster than (+)-borneol, especially since the oxidation of
(+)-isoborneol leads to (−)-camphor. Additionally, the specific activ-
ities of the Salvia officinalis L. enzymes towards the pure enantiomers
were compared to three previously characterized plant borneol dehy-
drogenases and one from Pseudomonas sp. TCU-HL1 (Table 2). The re-
actions were run in the same conditions as above with rac-1 and rac-2,
taking time-point measurements over 24 h for each enantiomer sepa-
rately. As previously reported, and also confirmed during our study, the
other dehydrogenases converted both (+)-borneol and (−)-borneol
with low specificity towards (+)-borneol. The highest selectivity was
found for ADH2 from Artemisia annua (Table 2), with a relative ratio of
reaction rates of 10 (Table 2, entry 3). Regarding the choice of nicoti-
namide cofactor, a strong preference for NAD+ was observed, in fact
when it was replaced by NADP+, activity was no longer observed (Fig.
S2). The same behavior was reported for LiBDH and ADH2, however,
this is different for the AaBDH from Artemisia annua, where both ni-
cotinamide cofactors are accepted-albeit NAD+ at a higher rate. Some
difference was noted in the selectivity of AaBDH during kinetic re-
solution towards rac-borneol, the conversion of pure enantiomers was
noted and attributed to the fact that measurements using pure
3

I. Drienovská, et al.
Phytochemistry172(2020)112227
Fig. 4. A) Selective oxidative kinetic resolution of
borneol enantiomers (+)-1 and (−)-1 and B) iso-
borneol enantiomers (+)-2 and (−)-2 to corre-
sponding camphor enantiomers 3. The selective oxi-
dation of (+)-borneol ((+)-1) leads to (+)-camphor
((+)-3), while selective oxidation of (+)-isoborneol
leads to (−)-camphor (−)-3).
Table 1
dehydrogenase activity was assayed at pH values ranging from 6.0 to
11.0. The optimal pH of the enzyme was determined to be in the al-
kaline range with maximum activity observed at a pH of 9.5. For the
optimum temperature, the activity of borneol dehydrogenase was
measured under standard assay conditions with the temperatures varied
between 25 and 70 °C; the optimal temperature for the initial rate of the
enzyme was determined to be 60 °C (Fig. S3).
Furthermore, kinetic parameters were determined for the oxidation
of (+)-borneol catalyzed by SoBDH2 (Fig. 5). The analysis was per-
formed by monitoring the formation of product (+)-camphor over time
Kinetic resolution of rac-borneol and rac-isoborneol catalyzed by alcohol de-
hydrogenases (2 mM final concentration of rac-1 or rac-2).
c[a]
(%)
E[b]
[a]
[a]
Entry
Enzyme
Subs.
Time (h)
ees
(%)
eep
(%)
1
2
3
4
5
6
LiBDH
AaBDH
ADH2
PsBDH
SoBDH1
SoBDH2
rac-1
rac-1
rac-1
rac-1
rac-1
rac-1
4
4
0.5
0.5
24
4
1
1
83
50
18
49
2
11
46
17
8
8
65
74
22
35
1.1
1.3
6.6
2.1
> 200
> 200
> 99
> 99
as
(0.05–2 mM) as well as cofactor NAD+ (10–400 μM). The parameters
obtained for (+)-borneol were quantified revealing KM of
0.16 0.033 mM, a kcat of 0.300
0.007 min−1 and a catalytic
efficiency of (kcat/KM) of 1871.1 M−1 min−1, respectively. The para-
meters obtained for NAD+ were KM of 0.048
0.007 mM, a kcat of
0.292
0.010 min−1 and a catalytic efficiency of 6104.2 M−1 min−1
a function of the concentration of the substrate (+)-borneol
7
8
9
10
11
12
LiBDH
AaBDH
ADH2
PsBDH
SoBDH1
SoBDH2
rac-2
rac-2
rac-2
rac-2
rac-2
rac-2
4
–
0.5
0.5
24
4
9
–
99
27
30
99
99
–
4
4
99
13
12
–
90
88
28
87
> 200
–
3
1.3
> 200
4.6
a
.
[a]
The KM value of SoBDH2 for (+)-borneol is slightly higher (160 μM),
albeit still very similar to other reported plant borneol dehydrogenses
(48.3 and 53.6 μM). The kcat and kcat/KM values are also comparable
with the reported values of Artemisia annua and Lavandula intermedia.
Microbial BDH is significantly more active, although KM is the highest
out of these enzyme (reported kinetic data of other ADHs, Table S2).
The comparatively low maximal reaction rates appear to suffice for
their role in the biosynthesis of camphor. Comparing SoBDH2 with
borneol dehydrogenase from sage leaf (Croteau et al., 1978) reveals
similarities in size, 124.8 kDa (as determined by size-exclusion chro-
matography, Fig. S4) compared to 91 kDa, suggesting the tetrameric
structure for both enzymes. Furthermore, both enzymes showed similar
alkaline pH optimum, which is unsurprising giving their similarity to
many alcohol dehydrogenases of plant origin. Interestingly, the ap-
parent KM, for (+)-borneol in case of SoBDH2 was slightly higher
160 μM–30 μM, respectively.
Determined by chiral GC analysis.
According to Chen et al. (1982)
[b]
Table 2
Specific activity towards (+)-borneol and (−)-borneol (2 mM final con-
centration).
Entry
Enzyme
vo_(+)-1a
vo_(−)-1a
v_(+)-1/v_(−)-1b
(μM.min−1
)
(μM.min−1
)
1
2
3
4
5
6
LiBDH
AaBDH
ADH2
PsBDH
SoBDH1
SoBDH2
0.06
0.20
12.77
10.89
0.94
1.81
0.01
0.08
1.28
6.31
0
6.0
2.5
10.0
1.7
> 100
> 100
0
[a]
Determined by chiral GC analysis.
Finally, the two new dehydrogenases were characterized in view of
potential synthetic applications. Racemic camphor and optically pure
(+)-camphor are readily available; while (−)-camphor is more difficult
to produce. Chemical reduction of rac-camphor leads to the production
of all 4 isomers, that is rac-1 and rac-2; depending on the type of che-
mical reduction, the ratio of this mixture is different (Brown and
Muzzio, 1966). The reduction using NABH4 gives approximately 4:1
ratio of rac-isoborneol to rac-borneol while the reduction of (+)-cam-
phor results in a mixture of (+)-borneol and (−)-isoborneol in a similar
ratio (Fig. 6). The fact that SoBDH1 selectively oxidizes (+)-borneol
but does not accept (−)-isoborneol allows for the selective conversion
enantiomers and substrate concentrations above the KM-value do not
take into account the competition between both enantiomers for the
active site, which depends on the difference in KM-values .
Both SoBDH1 and SoBDH2 were further examined for their catalytic
properties. However, the low stability of SoBDH1 above 20 °C and its
fast precipitation in different buffer conditions did not allow for full
kinetic analysis. This was, therefore, only undertaken with the second
enzyme, SoBDH2. The effect of pH on the activity of the purified
SoBDH2 (3.75 μM final concentration) was studied with
1 mM
(+)-borneol and 0.1 mM NAD+ as substrates at 25 °C and borneol
4

I. Drienovská, et al.
Phytochemistry172(2020)112227
Fig. 5. Kinetic characterization of the oxidation reaction of (+)-borneol catalyzed by SoBDH2. The lines represent the fit obtained using the Michaelis-Menten
equation constructed using Origin 2017 software.
of the former to obtain the unreacted (−)-isoborneol in pure form
(Fig. 7D). Interestingly, SoBDH2 converts three out of the four com-
pounds. From a mixture of all four diastereomers, (−)-borneol was left
unreacted (Fig. 7C). Using SoBDH1 with the same mixture, the oxida-
tion results in the formation of (+)-camphor and (+)-borneol. The
Salvia officinalis L. dehydrogenase, SoBDH2, thus appears to be very
promising catalysts for the synthesis of optically pure (−)-borneol
using racemic camphor as a starting material. Yet, the very low pro-
ductivities of all borneol dehydrogenases from plants characterized so
far necessitates substantial improvements in enzyme production yield,
specific activity and stability.
3. Conclusions
The recombinant production of the two borneol dehydrogenases
confirmed the pioneering work of the Croteau group that indicated the
presence of enantioselective enzymes in sage. The relatively close re-
lationship with around 50% sequence identity to unselective enzymes
isolated from Artemisia annua and Lavandula intermedia suggests that
these enzymes share a common ancestor whose selectivity remains
unknown. The fact that enantioselectivity is not necessary for the bio-
synthesis of (+)-camphor raises the question of which evolutionary
advantage, at least, two enantioselective dehydrogenases provide for
sage. The high enantioselectivity of both dehydrogenases and the
Fig. 6. A) Chemical reduction of racemic camphor leads to a mixture of four stereoisomers, from which SoBDH2 oxidizes three; B) Chemical reduction of
(+)-camphor produces a mixture of (+)-1 (20%) and (−)-2 (80%), from which SoBDH1 oxidizes (+)-1 and leaves (−)-2 unreacted.
5

I. Drienovská, et al.
Phytochemistry172(2020)112227
Fig. 7. Enzymatic oxidation of different mixtures of enantiomers of 1 and 2. A) Oxidation of rac-1 after 24 h catalyzed by SoBDH1/SoBDH2; B) Oxidation of rac-2
after 24 h catalyzed by SoBDH1/SoBDH2. C) Oxidation of a mixture of rac-1 and rac-2 by SoBDH2. D) Oxidation reaction of a mixture of (+)-1 and (−)-2 (stemming
from chemical reduction) of (+)-3) after 40 h.
surprising fact that they prefer (+)-borneol, and (+)-isoborneol, reveal
the possibility to use them for the catalytic separation of the en-
antiomers for the synthesis of either pure (−)-borneol or (−)-iso-
borneol. After chemical reduction of racemic camphor, SoBDH2 (being
selective towards borneol, but not isoborneol), allows for oxidation of
three isomers and leaves (−)-borneol unreacted. After reduction of
(+)-camphor, SoBDH1 converts (+)-borneol leaving (−)-isoborneol
unreacted. Both dehydrogenases thus represent a very useful addition
to the catalytic toolbox for the synthesis of bioactive terpenoids.
Furthermore, borneol dehydrogenases seem to be rather non-specific
enzymes, exhibiting a larger substrate scope and therefore allowing the
use of here-in reported enzymes for the functionalization of other
substrates as well.
4.2. Protein expression and purification conditions
Lysogeny broth (LB) media (Trypton 10 g/L, Yeast extract 5 g/L,
NaCl 5 g/L) (Carl Roth, Austria) was used for routine culturing of E. coli
with vigorous shaking at 37 °C and for expression. For the expression of
these enzymes in E. coli BL21(DE3), 2 mL of overnight culture, 500 mL
of LB media, 500 μL of ampicillin (100 μg/mL final concentration) or
kanamycin (40 μg/mL final concentration) were added in 2 L baffled
Erlenmeyer flasks which were incubated at 37 °C and 130 rpm in a
shaking incubator until an OD600 of 0.5–0.6 was reached. Subsequently,
protein production was induced with 500 μL IPTG (1 mM final con-
centration) and incubated overnight at 20 °C (LiBDH, AaBDH, PsBDH,
SoBDH1) or 28 °C (ADH2 and SoBDH2), both at 130 rpm. The cells
were harvested at 5500 rpm for 20 min at 4 °C. Pellets were re-
suspended in 20 mL of 100 mM potassium phosphate buffer (pH 8.0)
containing 10% glycerol, 1 mM DTT and 500 mM NaCl. Resuspended
cells (20 mL) were sonicated (6 min, 80% duty cycle, microtip limit 7)
and centrifuged (15,000 rpm, 60 min, 4 °C). The resultant supernatant
was collected and subsequently filtrated with 0.45 μM filters and di-
rectly used for catalysis.
Furthermore, filtrated supernatants of SoBDH1 and SoBDH2 were
used for purification using his-tag affinity chromatography. For pur-
ification, columns were washed with distilled water and equilibrated
with 100 mM potassium phosphate buffer (pH 8.0) containing 10%
glycerol, 1 mM DTT, 0.5 M NaCl and 30 mM imidazole. Elution was
carried out with 100 mM potassium phosphate buffer (pH 8.0) con-
taining 10% glycerol, 1 mM DTT, 500 mM NaCl and 500 mM imidazole.
Elution fractions containing pure protein were pooled together and
enzymes were rebuffered with 100 mM Tris-HCl buffer (pH 9.0) prior
kinetic analysis. Following the purification, the fractions were analyzed
4. Experimental
4.1. Materials
All chemicals were purchased from Sigma Aldrich and used without
further purification, unless otherwise stated. Codon-optimized synthetic
genes cloned into pET15b or pET41b(+) with sequences containing N-
or C- terminal polyhistidine tag (based on the suggestion of particular
variant from the literature) were ordered from GenScript, USA. A
summary of information regarding plasmids used in this study is given
in Table S3. E. coli BL21(DE3) was used for expression. Ni Sepharose 6
Fast flow column (GE Healthcare, Sweden) was used for purification.
Concentrations of DNA and protein solutions were determined based on
the absorption at 260 nm or 280 nm on a Thermo Scientific Nanodrop
2000 UV–Vis spectrophotometer. Enzymatic assays were recorded on
an Eon Microplate Spectrophotometer (BioTek, USA).
6

I. Drienovská, et al.
Phytochemistry172(2020)112227
by SDS-PAGE electrophoresis on 12% polyacrylamide gels
(ExpressPlus™ PAGE Gel, Genscript, USA) followed by Coomassie
staining. Protein concentration was determined by measuring absor-
bance at 280 nm on a Thermo Scientific Nanodrop 2000 UV–Vis spec-
trophotometer using the extinction coefficient of enzymes at 280 nm,
calculated using the Expasy ProtParam tool (https://web.expasy.org/
protparam). ε280 of SoBDH1 is 9190 M−1 cm−1 (for monomer) and ε280
of SoBDH2 is 10,345 M−1 cm−1 (for monomer) and 41,380 M−1 cm−1
(tetramer), respectively.
Declaration of competing interest
There are no conflicts to declare.
Acknowledgements
The authors acknowledge financial support by German Federal
Ministry of Education and Research (BMBF) for the project CbP-cam-
phor based polymers within the bio-economy international program
(grant No. 031B0501). We thank Dr. Gerhard Thallinger (TU Graz) for
discussions on phylogenetic relationships. We thank the 1000 Plants
(1 KP) initiative for the Salvia officinalis L. transcriptome data and
especially to Prof. Michael Deyholos for additional details about the
Salvia plant.
4.3. Size-exclusion chromatography
Analytical size-exclusion chromatography was carried out using a
HiLoad™ 16/60 Superdex™ 200 size-exclusion column (GE Healthcare).
Purified enzyme solutions (500 μL) were injected and separated at a
flow rate of 1 mL/min with 100 mM Tris-HCl, 500 mM NaCl buffer (pH
8.0). The column was calibrated using the standard Gel Filtration
Calibration Kit (Gel Filtration Cal Kit High Molecular Weight, GE
Healthcare). The molecular mass standards used were Ovalbumin
(44 kDa), Conalbumin (75 kDa) Aldolase (158 kDa) and Ferritin
(440 kDa).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112227.
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8