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R. Suthangkornkul et al. / Phytochemistry xxx (2016) xxx–xxx
b-glucosidases; therefore, the structural elements responsible for
substrate specificity in SBgl3 and other GH3 b-glucosidases need
to be investigated, as well as the physiological roles of the SBgl3
enzyme and substrate combination. Although there have been several
reports on the molecular characterization of two enzymes isolated
from oat and C. speciosus that hydrolyze furostanol glycoside 26-O-
b-glucosides, they are classified as GH1 b-glucosidases. Nonetheless,
this study focused on the Bgl3 gene and protein in only one species,
S. torvum; therefore, enzyme–substrate combinations need to be
investigated in other species of the Solanum genus. To our knowl-
edge, this is the first report on the expression and characterization
of a plant GH3 b-glucosidase that hydrolyzes furostanol glycoside
b-glucosidases was performed using the Clustal X program and
manually adjusted using the GenDoc program.
4.2. Construction of the recombinant plasmid and transformation into
P. pastoris
Using the pBluescript II/SBgl3 plasmid as a template, the mature
SBgl3 gene with and without a polyhistidine tag was PCR-amplified
using the SBgl3-F-1 primer containing a XhoI site and either the
SBgl3-R-1 or SBgl3-R-His primer, which both contained XbaI sites.
After digestion with XhoI and XbaI, the PCR product was cloned
2
6-O-b-glucosides.
into the vector pPICZ
by DNA sequencing carried out by Macrogen and designated
pPICZ A/SBgl3His. The pPICZ A/SBgl3His plasmid was linearized
aA (Invitrogen). The construct was confirmed
a
a
with SacI and transformed into P. pastoris GS115 by electropora-
4
. Experimental
tion. Positive transformants harboring the SBgl3 gene were selected
on YPDS plates containing 100 lg/ml zeocin.
4.1. Cloning of the full-length cDNA of the SBgl3 gene by the RACE
method
4.3. Expression of the recombinant SBgl3 in P. pastoris
All primers used in this study are shown in Supplementary
Table 1, unless stated. Total RNA was extracted from various
organs (young leaves, old leaves, germinated seeds, fruits, roots
and flowers) of S. torvum using the PureLink Plant RNA Reagent
kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions. Firstly, total RNA extracted from young leaves was
used as a template for synthesis of a partial cDNA using a Super-
Script III RT/Platinum Taq DNA polymerase Reverse Transcriptase
one step RT-PCR kit (Invitrogen) with the forward degenerate pri-
The selected transformant showing the highest activity was cul-
tured in BMGY medium at 30 °C until the OD600 reached 5.0. The
cells were harvested and resuspended in BMMY medium. The cul-
ture was incubated at 16 °C and MeOH was added at a 0.5% final
concentration every 24 h to maintain induction. After induction
for 120 h at 16 °C, the culture was harvested to obtain the cell-free
supernatant (CFS) for the purification of rSBgl3His.
0
0
mer 5 -GGVGCTGCMACNGCDCTMGARGTYAG-3 and reverse
0
0
degenerate primer 5 -ATBCKRCTCATDGGRATDATRTTRTG-3 (R, H,
B, and Y indicate degenerate sites: R, A/G; H, A/C/T; and Y, C/T),
which were designed from two internal peptide sequences, R(I/L)
GAATA(I/L)EVR and HN(I/L)(I/L)PMSR, from GH3 b-glucosidase
4.4. Enzyme assay and protein determination
Two assays for b-glucosidase were performed. The pNP-gly-
coside assay was used to study the sugar specificity of glycosides
as described previously, and the glucose oxidase/peroxidase assay
was performed as described previously (Arthan et al., 2006). One
nkat of enzyme activity was defined as the amount of enzyme
releasing 1 nmole of pNP or glucose from the substrate per second
at 37 °C, pH 5.0. Protein concentration was determined with the
Bio-Rad protein assay kit (Bio-Rad) using BSA as a standard or
the absorbance at 280 nm.
(Arthan et al., 2006). The PCR-amplified products were cloned into
the pGEMT easy vector and sequenced. The DNA sequence of the
SBgl3 gene fragment was used to design gene specific primers for
0
0
RACE. The 5 and 3 RACE kit (Invitrogen) was employed to deter-
mine the full-length cDNA following the manufacturer’s instruc-
0
tions. To perform 3 -RACE, the first-strand cDNA was synthesized
by SuperScript III RT using the GeneRacer oligo-dT primer. Gene-
0
0
specific primers named 3 _RACE_INS and 3 -GeneRACER were used
0
to amplify the 3 -end fragment of the SBgl3 gene.
0
To perform 5 -RACE, total RNA was dephosphorylated and then
4.5. Purification of the recombinant SBgl3His
ligated with GeneRacer RNA oligo according to the Invitrogen pro-
tocol. Total RNA ligated with GeneRacer RNA was then used as a
template for first-stand cDNA synthesis with the 5_RACE_SBgl3_-
out primer and SuperScript III RT. To determine the 5 -end of the
SBgl3 gene, the first-strand cDNA synthesized by SuperScript III
The CFS containing rSBgl3His was dialyzed against 20 mM
sodium phosphate buffer, pH 7.4. The dialysate was then loaded
onto a DEAE-cellulose column (2.5 ꢂ 5 cm) equilibrated with
20 mM sodium phosphate buffer, pH 7.4. The column was washed
with a 10-column volume of 20 mM sodium phosphate buffer, pH
7.4. The flow through fractions showing rSBgl3His activity were
collected, pooled, and dialyzed against 20 mM potassium phos-
0
RT with the GeneRacer oligo-dT primer, together with the
0
5
_RACE_SBgl3_out and GeneRacer 5 primers, were used for the
0
first PCR amplification to obtain the 5 -end fragment of the SBgl3
gene. The 5-RACE_SBgl3_ins and GeneRacer nested 5 primers were
0
2+
phate, pH 7.4. The dialysate was then loaded into a Ni -Sepharose
used for nested PCR amplification. The PCR-amplified product was
then cloned into the pGEMT vector (Promega, USA), transformed
FF column (1 ꢂ 4 cm). The elution was done with a stepwise gradi-
ent of imidazole including 300 and 500 mM imidazole in 20 mM
sodium phosphate buffer, pH 7.4. The active fractions containing
into E. coli DH5a, and DNA sequencing was carried out by the
2
+
Macrogen company.
rSBgl3His from the Ni -Sepharose FF step were pooled and con-
centrated using a Centricon filter with a molecular weight cutoff
of 10 kDa. The concentrated pools were run on a Sephacryl S-200
equilibrated with 20 mM sodium phosphate buffer, pH 7.4 contain-
ing 0.2 M NaCl. All fractions obtained from each step were assayed
for SBgl3His activity and protein concentration with a glucose oxi-
dase kit assay using torvoside A(1) as a substrate and by measuring
the absorbance at 280 nm, respectively.
0
0
According to the 3 - and 5 -RACE results, GH3_fl_forward and
GH3_fl_reverse primers were designed from the SBgl3 gene
sequence obtained by the RACE method and used to amplify the
full-length SBgl3 gene. The PCR-amplified product was cloned into
the EcoRV site of pBlueScript II SK (Stratagene), designated pBlue-
script II SK-SBgl3, and DNA sequencing was carried out by Macro-
gen. Alignment of SBgl3 with other structurally related GH3
Suthangkornkul, Rungarun
