Expression, purification and characterization of recombinant (E)-β-farnesene synthase from Artemisia annua

Article abstract of DOI:10.1016/j.phytochem.2005.03.027

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k _cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10^-3 s^-1, respectively resulting in the efficiency 4.5 × 10^-3 M^-1 s^-1. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni ²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg ²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.

Full text of DOI:10.1016/j.phytochem.2005.03.027

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 961–967
www.elsevier.com/locate/phytochem
Expression, purification and characterization of recombinant
q
(E)-b-farnesene synthase from Artemisia annua
*
Sarah Picaud, Maria Brodelius, Peter E. Brodelius
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182, Kalmar, Sweden
Received 14 March 2005; received in revised form 23 March 2005
Available online 3 May 2005
Abstract
A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-b-farnesene synthase, has been iso-
lated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated
pI = 5.03. The deduced amino acid sequence is 30–50% identical with sequences of other sesquiterpene synthases from angiosperms.
The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, b-farnesene, from farnesyl
diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the Km- and kcat-values for farnesyl diphosphate,
is 2.1 lM and 9.5 · 10ꢀ3
s
ꢀ1, respectively resulting in the efficiency 4.5 · 10ꢀ3
M
ꢀ1 sꢀ1. The enzyme exhibits substantial activity
in the presence of Mg2+, Mn2+ or Co2+ but essentially no activity when Zn2+, Ni2+ or Cu2+ is used as cofactor. The concentration
required for maximum activity are estimated to 5 mM, 0.5 mM and <10 lM for Mg2+, Co2+ or Mn2+, respectively. Geranyl diphos-
phate is not a substrate for the recombinant enzyme.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Artemisia annua; b-Farnesene synthase; cDNA cloning; GC–MS; Recombinant expression; Sesquiterpenes
1. Introduction
taken with A. annua. A number of sesquiterpenoids have
been isolated from leaves of A. annua. a-Copaene, b-
caryophyllene, trans-b-farnesene, muurola-4,11-diene,
selina-4,11-diene, germacrene D, b-selinene, bicycloger-
macrene, germacrene A, c-cadinene, a-humulene, and
amorpha-4,11-diene were identified in solvent extracts
of greenhouse-grown A. annua leaves by GC–MS (Bou-
wmeester et al., 1999). The olefinic sesquiterpenoids iso-
lated from A. annua represent different structural classes
and a number of sesquiterpene synthases catalyzing the
formation of the various carbon skeletons must be ex-
pressed in the plant.
Searches by us and other groups have revealed a
number of cDNAs from A. annua that encode different
sesquiterpene synthases (Fig. 1). The first cDNA clone
encoding a sesquiterpene synthase from A. annua re-
ported was that for epi-cedrol synthase (Mercke et al.,
The aerial parts of the plant Artemisia annua L.
(Asteraceae) (sweet wormwood) has during centuries
been used in traditional Chinese medicine (qinghao) to
treat various fever diseases. The plant produces the
endoperoxide sesquiterpene lactone antimalarial drug
artemisinin (qinghaosu). Following the discovery of
artemisinin, much phytochemical work has been under-
Abbreviations: b-FS, b-farnesene synthase; FPP, farnesyl diphos-
phate; GPP, geranyl diphosphate; IMAC, immobilized metal affinity
chromatography; IPTG, isopropyl thio-b-D-thiogalactoside.
q
The sequence reported in this paper has been deposited in the
GenBank Accession No. AY835398.
*
Corresponding author. Tel.: +46 480 447358; fax: +46 480 446262.
E-mail address: peter.brodelius@hik.se (P.E. Brodelius).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.03.027

962
S. Picaud et al. / Phytochemistry 66 (2005) 961–967
modification, provide a valuable addition to the arsenal
of natural compounds active in host plant resistance.
OH
8-epi-cedrol
2. Results and discussion
2.1. cDNA isolation and sequence analysis
1
Based on the sequence of a putative sesquiterpene
synthase from A. annua (van Geldre et al., 2000), a
1.8-kb fragment was amplified by PCR and cloned.
The full-length clone contained 1746 bp and an open
reading frame of 574 amino acids. The deduced mass
of the encoded protein is 66.9 kDa and the pI is cal-
culated to be 5.03. The low pI and the molecular
weight are similar to those reported for other plant
sesquiterpene synthases. The isolated clone (GenBank
Accession No. AY835398) encodes (E)-b-FS as deter-
mined below.
2
4
OPP
amorpha-4,11-diene
farnesyl diphosphate
β-farnesene
3
A comparison of the nucleotide sequence obtained
with that of the putative sesquiterpene synthase (Gen-
Bank Accession No. AJ271793) (van Geldre et al.,
2000) revealed some differences. The deduced amino
acid sequences of the two sequences differ in the overall
length by three amino acids. Alignment of the two de-
duced amino acid sequences to those of the other three
sesquiterpene synthases from A. annua shows that the
shorter sequence obtained in this study appears to be
correct due to better alignment and higher homology.
From this alignment it is also evident that the b-FS
has an extension in the N-terminal as compared to the
other Artemisia sesquiterpene synthases. This extra se-
quence of 22 residues contains a number of hydroxyl-
ated amino acids and it is evident that this extra
sequence is highly hydrophilic. According to an iPSORT
prediction (http://biocaml.org/ipsort/iPSORT/) this ex-
tra sequence does not carry a signal or sorting sequence
(Bannai et al., 2002).
The deduced amino acid sequence of b-FS from A.
annua shows relatively high identity/similarity to other
sesquiterpene synthases and especially to those from
A. annua. It is interesting to note that the b-FS from
A. annua exhibits a considerably higher homology to
other sesquiterpene synthases from this plant (the four
Artemisia sesquiterpene synthases show an identity/sim-
ilarity between 47/65% and 59/78% to each other) than
to b-FSs from other plants (the Artemisia FS shows an
identity/similarity of 42/61%, 33/56% and 21/39% to
the b-FSs from Citrus junos, Mentha · piperita and Z.
mays, respectively). Obviously, the level of homology
between deduced amino acid sequences of synthases
showing identical substrate/product specificity (in this
case b-FSs) from different species can be lower than
the homology between synthases catalyzing the forma-
tion of completely different terpene structural types from
the same plant.
β-caryophyllene
Fig. 1. Reactions catalyzed by the four sesquiterpene synthases cloned
from Artemisia annua: 1, epi-cedrol synthase (ECS); 2, amorpha-4,11-
diene synthase (ADS); 3, b-caryophyllene synthase (CS); 4, (E)-b-
farnesene synthase (FS).
1999; Hua and Matsuda, 1999). A number of groups
have reported the molecular cloning of amorpha-4,11-
diene synthase (Mercke et al., 2000; Chang et al.,
2000; Wallaart et al., 2001). This enzyme is responsible
for catalyzing the committed step in the biosynthesis
of artemisinin (Bouwmeester et al., 1999). A third
cDNA encoding b-caryophyllene synthase has been de-
scribed (Cai et al., 2002). Van Geldre et al. (2000) re-
ported on the cloning of two putative sesquiterpene
synthases from A. annua. One of these clones (GenBank
Accession No. AJ271972) corresponds to the b-caryo-
phyllene synthase (Cai et al., 2002) while the product
selectivity of the other clone (GenBank Accession No.
AJ271973) has remained unknown. We have now re-
cloned this sesquiterpene synthase (GenBank Accession
No. AY835398) and report that it encodes (E)-b-farne-
sene synthase (b-FS), which converts farnesyl diphos-
phate to the acyclic sesquiterpene (E)-b-farnesene (Fig.
1). (E)-b-Farnesene occurs in a wide range of both plant
and animal taxa. It is extensively used as a semiochem-
ical in insects and plants. During recent years cDNA
clones encoding (E)-b-FS has been isolated from pep-
permint (Mentha · piperita) (Crock et al., 1997), Citrus
junos (Maruyama et al., 2001) and Zea mays (Schnee
et al., 2002). The enzymes from peppermint and C. junos
produce essentially one product from FPP while the
maize enzyme produced three sesquiterpenoids: (E)-b-
farnesene (26%), (E)-b-nerolidol (29%) and (E,E)-farne-
sol (45%). (E)-b-FS cDNA clone(s) may, by transgenic

S. Picaud et al. / Phytochemistry 66 (2005) 961–967
963
2.2. Bacterial expression
2
100
(a)
Production of recombinant protein was carried out
under standard conditions using Escherichia coli
BL21(DE3) cells transformed with the bacterial expres-
sion vector pET28 carrying the b-FS cDNA. SDS–
PAGE revealed an IPTG inducible band at around 65
kDa. The apparent molecular mass is somewhat smaller
than the calculated size (69.1) for the fusion protein
composed of the b-FS and the histidine leader peptide
derived from pET28.
Crude extracts of E. coli expressing the cloned sesqui-
terpene synthase displayed a high activity with [1-3H]-
FPP as substrate. GC–MS analysis of the enzymatic
product revealed the formation of a single sesquiterpe-
noid (peak 2 in Fig. 2(a)). Comparison of the MS-spec-
trum of this product (Fig. 2(b)) to spectra of the parent
compound reveals that the enzyme catalyzes the forma-
tion of (E)-b-farnesene (Fig. 2(c)).
80
60
40
1
3
20
0
6
8
10
12
14
retention time (min)
69
100
80
(b)
93
peak 2
R = 11.42 min
f
60
41
133
2.3. Purification of recombinant b-farnesene synthase
40
79
120
161
20
0
In order to facilitate an easy purification of the b-FS,
the enzyme was expressed in fusion with a N-terminal
affinity tag using the bacterial expression vector
pET28. This vector encodes a hexa-histidyl leader se-
quence under the control of the T7 RNA polymerase
promoter. The enzyme is extended with 20 amino acids
after fusion to the His6-tag.
Recombinant b-FS was purified by immobilized
metal affinity chromatography (IMAC) on a HiTrap
Chelating HP column loaded with Co2+. From SDS–
PAGE it is evident that a relatively efficient purification
of the recombinant enzyme is achieved after the IMAC
(data not shown). Western blotting experiments using
rabbit polyclonal amorpha-4,11-diene synthase antibod-
ies showed that the purified b-FS cross-reacts with these
antibodies (data not shown).
55
107
204
189
147
40
60
80
100 120 140 160 180 200 220
M/Z
69
100
80
(c)
(Ε ) β-farnesene
R = 11.42 min
f
93
41
60
40
133
79
146
120
161
55
20
0
107
204
189
40
60
80
100
140 160 180 200 220
120
M/Z
Fig. 2. GC–MS analysis of the sesquiterpene product of the
recombinant enzyme: (a) GC elution profile of the olefinic products
generated by from FPP by the recombinant enzyme. Peak 1 and peak 3
are not sesquiterpenoids; (b) mass spectrum of the major biosynthetic
product (Rf = 11.42) (peak 2); (c) mass spectrum of trans-b-farnesene
(Rf = 11.42).
2.4. pH-Optimum of b-farnesene synthase
The recombinant purified b-FS exhibited maximum
activity at around pH 6.5 to 7.0 (data not shown). Many
plant sesquiterpene synthases exhibit maximum activity
in the pH range of 6.5–7.5. Plant sesquiterpene syn-
thases are generally fully active at neutral pH values.
This is in line with the assumption that they are localized
to the cytosol of the cell.
activity is observed in the presence of Mg2+ or Co2+
.
In the presence of Mn2+, Ni2+, Cu2+ or Zn2+ essentially
no activity is observed at the tested concentrations.
However, the b-FS activity is highly dependent on the
concentration of the ion used and at low concentrations
of Mn2+ the b-FS exhibits a relatively high activity (Fig.
3(d)). From Fig. 3(b) to (d), it is clear that maximum b-
FS activity is observed at different metal ion concentra-
tions. The concentration required for maximum activity
are estimated to 5 mM (Fig. 3(b)), 0.5 mM (Fig. 3(c))
and <10 lM (Fig. 3(d)) for Mg2+, Co2+ or Mn2+, respec-
tively. However, using Mn2+ or Co2+ as cofactor a
2.5. Divalent metal ion dependence of b-farnesene
synthase
Sesquiterpene synthases have an absolute require-
ment for a divalent metal ion as cofactor. The activity
of b-FS has been measured in the presence of a number
of different divalent metal ions (Fig. 3(a)). Substantial

964
S. Picaud et al. / Phytochemistry 66 (2005) 961–967
FPP in the presence of Mg2+ is in the same range as
400
300
200
(a)
0.5 mM
2.5 mM
5.0 mM
those for other plant sesquiterpene synthases. As re-
flected by the kcat-value, this enzyme is a relatively slow
enzyme, which is a general feature of terpene synthases.
Sesquiterpene synthases exhibit turnover numbers in the
range 0.01–0.3 sꢀ1 (Cane, 1990). Pre-steady-state kinetic
analysis on tobacco epi-aristolochene synthase (Mathis
et al., 1997) and trichodiene synthase (Cane et al.,
1997) implies that plant sesquiterpene synthases operate
by a rapid equilibration with FPP to form a precatalytic
enzyme–substrate complex. The next step is a slightly
slower conversion of FPP to hydrocarbons. The release
of products is most likely the rate-limiting step of the
reaction.
100
0
Co2+
Cu2+
Zn2+ Mn2+ Mg2+
Ni2+
400
300
200
100
(b)
2.7. Substrate specificity of b-farnesene synthase
In the presence of Mn2+ but not with Mg2+, GPP is a
substrate for different sesquiterpene synthases (Cai et al.,
2002; Colby et al., 1998; Ko¨llner et al., 2004; Mercke
et al., 1999; Picaud et al., 2004). However, the purified
recombinant (E)-b-FS from A. annua does not convert
GPP to product in the presence of Mg2+- or Mn2+-ions
at any of the concentrations tested (10 lM to 5 mM).
Mg2+
0
200
(c)
100
Co2+
0
200
Mn2+
100
0
2.8. Products formed by b-farnesene synthase
(d)
The recombinant (E)-b-FS produces a single sesqui-
terpene during catalysis as illustrated in Fig. 2(a). Many
recombinant sesquiterpene synthases give rise to more
than one product (Crock et al., 1997; Colby et al.,
1998; Mercke et al., 1999; Picaud et al., 2004; Steele
et al., 1998). Most often one major product and a num-
ber of by-products are obtained. Premature or alterna-
tive quenching of the reaction cascade may cause the
formation of by-products. Other a- and b-farnesene syn-
thases also show high product selectivity (Crock et al.,
1997; Martin et al., 2004; Mercke et al., 2004; Pechous
and Whitaker, 2004).
0
1
2
3
4
5
ion concentration (mM)
Fig. 3. Activity of b-FS in the presence of different divalent metal ions:
(a) b-FS activity at three different concentrations of divalent metal
ions; (b) b-FS activity as function of Mg2+ concentration; (c) b-FS
activity as function of Co2+ concentration; (d) b-FS activity as
function of Mn2+ concentration.
somewhat lower specific b-FS activity is observed than
in the presence of Mg2+. In addition to this, the enzyme
is strongly inhibited by increased concentrations of
Mn2+ at a relatively low concentration (>20 lM). Inhi-
bition by high Mn2+-concentrations has also been ob-
served for amorpha-4,11-diene synthase (Picaud et al.,
2004), and (E)-nerolidol synthase from maize (Degen-
hardt and Gershenzon, 2000). In a number of studies,
it has been shown that maximum enzyme activity is
reached at a considerable lower concentration of Mn2+
than Mg2+ for sesquiterpene synthases (Cai et al.,
2002; Schnee et al., 2002; Picaud et al., 2004) but in gen-
eral the specific activity is lower with Mn2+ as cofactor.
3. Experimental
3.1. Reagents
Restrictions enzymes SpeI, NheI and SacI and the T4
DNA ligase were purchased from New England Biolabs.
Pyrobestꢁ DNA polymerase and the corresponding buf-
fer were supplied by Takara Bio Inc. DNA BigDyee
Terminator Cycle Sequencing Kit was obtained from
Perkin–Elmer. HiTrap Chelating HP and PD-10 col-
umns Sephadexe G-25M were from Amersham-Phar-
macia Biotech. [1-3H]-FPP (0.59 TBq/mmol) was
purchased from Amersham-Pharmacia Biotech and
[1-3H]-GPP (0.56 Tbq/mmol) from American Radio-
chemical Co. IPTG was obtained from Saveen Werner
2.6. Steady-state kinetics of b-farnesene synthase
Steady-state kinetic parameters of the purified recom-
binant (E)-b-FS were determined at pH 7.0. The Km-va-
lue (2.1 lM), kcat-value (9.5 · 10ꢀ3
s
ꢀ1) and enzyme
efficiency (kcat/Km = 4.5 · 10ꢀ3
M
ꢀ1 sꢀ1) recorded for

S. Picaud et al. / Phytochemistry 66 (2005) 961–967
965
AB. FPP, GPP and kanamycin were obtained from Sig-
ma. All other biochemicals and reagents were purchased
from commercial sources.
according to standard procedures. A single colony was
used to inoculate 10 ml LB medium containing kanamy-
cin (50 lg/ml) at 37 ꢂC overnight. Two ml of this culture
were added to 200 ml of fresh LB containing kanamycin.
Bacteria were grown at 37 ꢂC to OD600 = 0.5. Induction
was achieved by addition of 0.2 mM IPTG. The cells
were maintained for 4 h at 30 ꢂC and then harvested
by centrifugation (2000g, 10 min). Subsequently, the
bacteria were suspended in half of the previous volume
in 20 mM sodium phosphate buffer pH 6.5 containing
10% (v/v) glycerol and 0.5 M NaCl. After a new centri-
fugation and re-suspension of the cells in 1/10 of the ori-
ginal culture volume, sonication of the cells was carried
out for 2 min in 5 s pulses with 5 s between pulses on ice
(Vibracell power sonicate setting 12– 15 W). The cell ly-
sate was then centrifuged at 10000g for 30 min at 4 ꢂC
and the supernatant, containing the soluble recombinant
enzyme was filtrated on a 0.45 lm non-pyrogenic sterile
filter.
3.2. Plant growth
Plants of A. annua were grown from seeds in a green
house at 20 ꢂC with a 16-h light period.
3.3. Cloning of the b-farnesene synthase cDNA by
RT-PCR
RNA from mature A. annua leaves harvested at flow-
ering stage was extracted with the Purescriptꢁ Total
RNA Isolation Kit (Gentra Systems) according to the
manual. First strand cDNA synthesis was initiated on
1.0 lg of total RNA at the poly(A)-tail of mRNA using
the adapter primer (AP) and the Superscript II reverse
transcriptase (Invitrogen). After synthesis of the first
cDNA strand, mRNA was destroyed with RNase H
(Invitrogen). The complete open reading frame was
amplified by PCR using the cDNA synthesized as tem-
plate and using specific primers designed from the se-
quence putative sesquiterpene synthase (GenBank
Accession No. AJ271793) (van Geldre et al., 2000).
The primers used were: for the forward primer
3.5. Purification of b-farnesene synthase
The cell lysate containing the soluble recombinant en-
zyme was passed through a 5 ml HiTrap Chelating HP
column (Amersham Biosciences) loaded with Co2+
.
After washing with 30 mM imidazole, bound enzyme
was eluted in 20 mM sodium phosphate buffer, pH 6.5,
containing 10% (v/v) glycerol, 0.5 M NaCl and 300
mM imidazole. Fractions (1 ml) were collected and frac-
tions containing activity were pooled and passed through
a PD-10 column (Amersham Biosciences) equilibrated
with 20 mM sodium phosphate buffer, pH 6.5, contain-
ing 10% (v/v) glycerol and 0.5 M NaCl to remove imid-
azole. The purified enzyme was frozen in aliquotes in
liquid nitrogen and store at ꢀ20 ꢂC until used.
50-gatactagtatgtcgactcttcctatttctag-30
including
the
restriction site SpeI (underlined) and the reverse primer
50-cagagagctcttagacaaccatagggtgaac-30 including the
restriction site SacI (underlined). The obtained DNA
fragment with a length of 1746 bp was electrophoresed
through a 1% agarose gel. After gel purification, the
PCR fragment was A-tailed using the Taq-polymerase
(Invitrogen) and cloned into the plasmid vector
pGEMT-Easy (Promega). The plasmid obtained was
transformed into competent E. coli NovaBlue cells
according to standard procedures. The cells were se-
lected on ampicillin plates (50 lg/ml) complemented
with X-Gal and IPTG. The resulting plasmid DNA
was sequenced using the BigDyee Terminator Cycle
Sequencing Kit for labeling of the sequencing reactions.
Collection of the sequence data was carried out on an
ABI PRISMe 310 Genetic Analyzer. Oligonucleotides
were synthesized according to the sequence information
and used directly as primers for further sequencing. The
cDNA fragment was excised from pGEMT-Easy vector
by restriction digestion with SpeI and SacI and trans-
ferred into the pET28 expression vector (Novagen) di-
gested by NheI and SacI in frame with an affinity tag
(His6). Transformants were selected on LB plates con-
taining kanamycin (50 lg/ml).
3.6. Electrophoresis and western blotting
SDS–PAGE electrophoresis was carried out using
MES-buffer on 4–20% Tris/glycine gels or on NuPAGE
4–12% Bis/Tris gels from Invitrogen according to
instructions supplied by the manufacturer using a No-
vex mini electrophoresis cell. After electrophoresis, the
gels were either stained with Coomassie blue or the pro-
teins transferred to nitrocellulose membranes using a
Mini Trans-Blot module (Novex) according to instruc-
tions supplied by the manufacturer. The correct transfer
was checked by including a mixture of prestained pro-
tein markers on the PAGE gel (Kaleidoscope Prestained
Standards; Bio-Rad). Rabbit polyclonal ADS antibod-
ies and goat-anti-rabbit conjugated with alkaline phos-
phatase were use as primary and secondary antibodies,
respectively. The blot was developed with the 5-bro-
mo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium
system according to the manufacturerÕs instructions
(Bio-Rad).
3.4. Bacterial expression of the b-farnesene synthase
The plasmid pET28 containing the cDNA fragment
was transformed into competent E. coli BL21(DE3) cells

966
S. Picaud et al. / Phytochemistry 66 (2005) 961–967
3.7. Standard enzyme assay
equal volumes of n-pentane (Sigma). The 4 fractions ob-
tained were pooled and the pentane phase was concen-
trated by evaporation under a stream of helium gas to
around 50 ll. The sample was analyzed by capillary
GC on an Agilent 6890 gas chromatograph equipped
with a HP-5M5 5% phenyl methyl silohexane capillary
column (0.25 lm film thickness, 0.25 mm i.d. · 30 m)
and an Agilent 5973 Network Mass Selective Detector.
The GC injection port was operated at 100 ꢂC. The oven
temperature was programmed from 100 to 200 ꢂC at 5
ꢂC minꢀ1 and from 200 to 250 ꢂC at the rate of 25
ꢂC minꢀ1. The final temperature was maintained for 26
min. Helium was used as a carrier gas and sample (1.0
ll) was injected in split mode. Mass spectra were re-
corded in electron impact mode at 70 eV with scanning
from m/z 34 to 400 at 3.93 scans sꢀ1. Sesquiterpenoids
were identified by comparing the mass spectra and
retention indices with those of spectra found in the Mass
Finder 2.1 database and other libraries.
An aliquot of bacterial extract (48 ll) or purified en-
zyme (10 ll) was assayed in 20 mM sodium phosphate
buffer, pH 6.5, containing 5 mM MgCl2, 10% glycerol
and 23 lM [1-3H]-FPP (1.4 MBq) in a total volume of
50 ll. After 20 min incubation at 30 ꢂC, the reactions
were stopped by addition of an equal volume of 0.2 M
KOH containing 0.1 M EDTA. Subsequently, the reac-
tion mixture was extracted with hexane (0.5 ml) and the
hexane extracts were passed over a small column filled
with 250 mg of silica (Merck; size: 0.2–0.5 lm) into a
scintillation vial. The column was rinsed with additional
hexane (2 · 1 ml), which was collected in the same scin-
tillation vial. Vials were examined for radioactivity by
scintillation counting on TRI-CARB 2100 TR Liquid
scintillation Analyser (Packard). Three replications were
run for each sample.
3.8. pH optimum determination
3.12. Protein concentrations
For characterization, the purified recombinant en-
zyme was used (see above). The pH optimum was deter-
mined in buffers of 50 mM Bis–Tris, 50 mM sodium
phosphate or 50 mM Tris–HCl containing 5 mM MgCl2
and 10% glycerol, in the pH range from 5.0 to 9.5 using
1.5 lg of the purified protein and 18.7 lM [1-3H]-FPP in
a final volume of 100 ll. Samples were incubated at 30 ꢂC
for 10 min.
Protein concentrations of extracts and purified re-
combinant b-FS were determined according to Bradford
(1976) using IgG as standard.
Acknowledgements
The financial support from University of Kalmar,
The Crafoord Foundation and the European Union
(Marie Curie Scholarship to S.P.) is acknowledged.
3.9. Kinetic analysis of the b-farnesene synthase
Kinetic analysis of enzymes was conducting using 1.5
lg of purified protein per assay (100 ll). The assays were
conducted according to the standard assay with 0.5, 1, 2,
5 or 10 lM [1-3H]-FPP. Km-constants were determined
from Lineweaver–Burk plots and kcat-values were esti-
mated from the protein concentration using a molecular
weight of 69.1 kDa (b-FS + affinity tag).
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