Catalysis of Transglycosylation by ꢀ-Glucosidases
Protein & Peptide Letters, 2013, Vol. 20, No. 1 103
Tmꢀgly were separately cultivated in 500 mL of YPD me-
dium (1% yeast extract, 2% peptone and 2% glucose; w/v)
for 18 h at 28 °C and 150 rpm. After that, cells were harvest
by centrifugation (3,000 xg and 4 °C for 5 min) and washed
with sterile water. Then those cells were cultivated in BMM
medium (1.34% yeast nitrogen base and 0.00004 % biotin
(w/v) in 100 mM potassium phosphate buffer pH 6) contain-
ing 1 % methanol (v/v) for 72 h at 28 °C and 150 rpm. The
supernatant was collected after centrifugation (3,000 xg and
4 °C for 5 min) and ꢀ-glucosidases were purified by sedi-
mentation in 0.69 g/mL (NH4)2SO4. The pellet containing
ꢀꢀglucosidases was ressuspended in 100 mM sodium citrate
– sodium phosphate buffer pH 6 and dialyzed against the
same buffer for 18 h at 4 °C. K201F mutant Sfꢀgly was pro-
duced in BL21(DE3) bacteria (Stratagene, La Jolla, CA,
USA) using vector pET46 (Novagen, Darmstadt, Germany)
and purified by affinity chromatography in His Trap columns
(GE HealthCare, Chalfont, St. Giles, UK) according previ-
ously described [13].
E + Glc
(P2)
H2O
hydrolysis
E + Glc-NP
(S1)
E-Glc
+
NP
transglycosylation
(P1)
Glc-NP
(S2)
E + Glc-Glc-NP
(P2)
Figure 1. Schematic view of hydrolysis and transglycosylation
reactions catalyzed by ꢀ-glucosidases. Free enzyme (E) binds the
substrate (p-nitrophenyl ꢁ-glucoside; Glc-NP; S1) forming the en-
zyme-substrate complex (not represented) which originates the
covalent intermediate (E-Glc; ꢁ-glycosyl-enzyme) with the con-
comitant release of the first product (P1). The attack of a water
molecule on this intermediate characterizes the hydrolysis (upper
route), whereas the attack by a second substrate (S2, also called
acceptor) defines the transglycosylation route (bottom route). Prod-
uct P1 is common to both routes, whereas glucose (Glc) is produced
only in the hydrolysis. This scheme is also valid for methylumbel-
liferyl ꢀ-glucoside as substrate.
Site-directed Mutagenesis Experiments
Site-directed mutagenesis experiments were performed
using
QuikChange
Site-Directed
Mutagenesis
kit
(Stratagene, La Jolla, CA, USA) according manufacturer
instructions.
Considering that for reactions with NPꢀglc and MUꢀglc
%H and %T represent respectively the fractions of a ꢀ-
glucosidase sample following the hydrolysis and transglyco-
sylation routes, thus %T + %H = 100 (equation 1).
Enzymatic Assays
Samples of purified ꢀ-glucosidases previously dialyzed
against 100 mM citrate-phosphate buffer pH 6 were incu-
bated at 30 °C with p-nitrophenyl ꢁ-glucoside (NPꢀglc) or
methylumbellyferyl ꢀ-glucoside (MUꢀglc) prepared in 100
mM sodium citrate-sodium phosphate buffer pH 6. Reactions
were interrupted by boiling (3 min) and split in two equal
aliquots, which were used to detect the products. In the case
of reactions with NPꢀglc, one of the samples was used to
detect p-nitrophenolate (NP) production by absorbance at
420 nm after addition of 250 mM sodium carbonate-sodium
bicarbonate buffer pH 11, whereas the second aliquot was
used to detect glucose (glc) by using a modified version of
the glucose oxidase method [14], in which 50% H2SO4 was
added to the sample previous to the determination of absor-
bance at 540 nm. In the case of reactions with MUꢀglc, the
formation of methylumbelliferone (MU) was detected by
fluorescence emission at 450 nm using excitation wavelength
of 360 nm after addition of 100 mM glycine-NH4OH buffer
pH 10.5, whereas glucose (glc) was detected using glucose
oxidase method [14].
As NP and MU are produced in both routes, whereas glc
is produced exclusively on the hydrolysis, their rates of pro-
duction are proportional to the fraction of enzymes engaged
on each route. Hence
vNP/vglc = (%T + %H) / %H (equation 2) and vMU/vglc = (%T
+ %H) / %H
(equation 3)
Therefore, the fraction of ꢀ-glucosidases engaged on the
hydrolysis (%H) is determined based on the vNP/vglc and
vMU/vglc, whereas %T corresponds to the complementary per-
centage.
% H = 100 / vNP/vglc (equation 4) and % H = 100 / vMU/vglc
(equation 5)
Hence the effect of the substrate concentration on the rate
of glucose production is described by the Michaelis-Menten
equation multiplied by %H, because this operation intro-
duces the availability of the ꢀ-glucosidase to catalyze the
hydrolysis for each substrate concentration.
vglc = (Vmax[S] / Km + [S]) . %H
(equation 6)
Determination of Transglycosylation to Hydrolysis Ra-
tios
Similarly, the effect of the substrate concentration on the
transglycosylation rate is represented by the rate equation for
a Ping-Pong mechanism multiplied by %T.
For reactions with NPꢀglc and MUꢀglc the first product,
NP and MU, respectively, is common to both routes. Glc is
released only in the hydrolysis, whereas it is incorporated in
an oligosaccharidic product in the transglycosylation (Fig.
1). Thus, production ratios of NP or MU to glc (vNP/vglc and
vMU/vglc) higher than 1 indicate that glc was incorporated in
the transglycosylation product. Additionally, as demon-
strated below, such experimental ratios allow the determina-
tion of the fraction of a ꢀ-glucosidase sample engaged on
each reaction route.
vransglycosylation = (Vmax[S1][S2] / Km2 + [S1] + Km1[S2] +
[S1][S2]) . %T (equation 7)
Hence the effect of the substrate concentration on the rate
of NP or MU production corresponds to the sum of equations
6 and 7.
vNP = [(Vmax[S]/Km + [S]) . %H] + [(Vmax[S1][S2]/Km2 + [S1] +
Km1[S2] + [S1][S2]) . %T] (equation 8)