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N. Bridiau, T. Maugard / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 24–31
5 or 50 mM of lactose or LacNAc were treated at 100 ◦C for 5 min
in water/t-BuOH (70:30, v/v) mixtures or in water, under stirring
conditions. They were then cooled down for 5 min in an ice bath
and analysed by NP-HPLC, showing that neither lactose nor LacNAc
underwent chemical hydrolysis in these experimental conditions.
The initial velocities were calculated from the linear relationship
of the lactose concentration or of the total concentration of galac-
tosylated derivatives of GlcNAc against reaction time (0–10 min).
For the product inhibition study, initial velocity measurements
of the GlcNAc transgalactosylation were performed using the same
procedure, except that the reaction mixtures (1.5 mL) were com-
posed of various amounts of oNPG (5–50 mM), GlcNAc (50 mM) and
oNP (0–5 mM).
compound after its complete solubilisation was then considered as
its maximal solubility.
3. Results and discussion
3.1. Effect of t-BuOH on the kinetic characterisation of the total
transformation of oNPG and lactose catalysed by BcˇGal in
aqueous solutions and water/t-BuOH (70:30, v/v) mixtures
The total transformation of oNPG, which occurs during the trans-
galactosylation of GlcNAc catalysed by BcˇGal, was monitored in
initial velocity experiments conducted in water/t-BuOH (70:30,
v/v) mixtures using various GlcNAc concentrations within a range
5–50 mM in order to evaluate the kinetic mechanism of the enzyme.
The reaction was also carried out in the absence of GlcNAc in
water, in 100 mM phosphate buffer, pH 6.5 (10 mM MgCl2), or in
water/t-BuOH (70:30, v/v) mixtures; the aim in every case was to
identify the kinetic constants for BcˇGal in water and water/t-BuOH
(70:30, v/v) mixtures. The total transformation of lactose catalysed
by BcˇGal was also studied in water and water/t-BuOH (70:30,
v/v) mixtures. The transformation of oNPG and lactose was moni-
tored through the release of o-nitrophenol and the disappearance
of lactose in the media, respectively.
Systematic analysis of the rates, which were dependent on the
GlcNAc was parametric. The results concerning the experiments
performed with oNPG as a galactosyl donor are shown in Fig. 1.
Kinetic constants determined for all experiments are presented
in Table 1. These results provide information about the effects of
t-BuOH on the catalytic activity of BcˇGal.
2.4. Data analysis
Except for the product inhibition study, all initial rate data were
fitted to the equation that describes the steady-state sequential
ordered Bi Bi mechanism equation (Eq. (1)) using nonlinear least-
squares regression analysis in the Enzyme Kinetics Module of Sigma
Plot, “Enzyme Kinetics 2004 1.2” (Systat Software Inc., San Jose,
USA).
Vmax[A][B]
KiaKmB + KmB[A] + KmA[B] + [A][B]
v =
Initial rate data corresponding to the product inhibition study were
fitted to all the inhibition models in the “Enzyme Kinetics 2004
1.2” software using nonlinear least-squares regression analysis. The
equation for full noncompetitive inhibition (Eq. (2)) was relevant
to the work presented here.
Vmax[A]
(KmA + [A])(1 + [I]/Ki)
v =
(2)
(1) First, it was found that the apparent initial velocity
A and B represent the substrates oNPG and GlcNAc, respectively.
KmA and KmB are the Michaelis constants for A and B, respectively.
Vmax is the maximum velocity. Kia is defined as the dissociation
constant of A from the binary complex E·A and Ki is the inhibition
constant for the inhibitor used [27].
(Vmax,app) of the total transformation of oNPG was about
2.5-fold higher in water (Vmax,app = 8967 mol min−1 mL−1
;
entry 6) than in
a
water/t-BuOH (70:30, v/v) mixture
entry 1). Thus t-BuOH
(Vmax,app = 3478 mol min−1 mL−1
;
seemed to negatively affect the catalytic activity of BcˇGal.
(2) Secondly, the Km,app of oNPG was found to increase from 9.0 mM
in water (entry 6) to 14.8 mM in a water/t-BuOH (70:30, v/v)
mixture (entry 1), which characterised a lack of affinity of
BcˇGal for oNPG in the presence of t-BuOH, most probably due
to the solvation of the enzyme by t-BuOH. This solvent might
indeed enter the catalytic site and sterically hinder oNPG. Log-
ically, the decrease in the Vmax,app associated with the increase
in the Km,app of oNPG observed when using t-BuOH resulted in
a higher catalytic efficiency in water than in a water/t-BuOH
(70:30, v/v) mixture.
(3) Thirdly, the effects of t-BuOH on the kinetic parameters of
the lactose transformation catalysed by BcˇGal were shown
to be slightly different. While the Vmax,app values in water
(entry 9) and in a water/t-BuOH (70:30, v/v) mixture (entry 8)
Km,app of lactose was found to be 36-fold higher in a water/t-
BuOH (70:30, v/v) mixture than in water. These results are
in accordance with those provided by the work of Hancock
et al. [28], which showed that the addition of acetonitrile as
used to transform p-nitrophenyl-ˇ-d-galactopyranoside, due to
the increase of Km in conjunction with the higher concentra-
tion of acetonitrile. Similar results were described by Takahashi
et al. [29] who studied the Rhizopus niveus glucoamylase-
catalysed hydrolysis of maltose in the absence or presence of
To visualise the quality of fitting, the experimental data points
are presented in plots along with theoretical lines fitted by “Enzyme
Kinetics 2004 1.2” software.
2.5. HPLC analyses
Structural and quantitative analyses of lactose or galactosy-
lated derivatives of GlcNAc were conducted using a LC/MS-ES
system from Agilent (1100 LC/MSD Trap mass spectrometer VL),
with an Uptisphere 6 DIOL normal-phase column (250 mm × 4 mm,
at room temperature and at a flow rate of 1 mL min−1. Lactose was
detected and quantified by differential refractometry while galac-
tosylated derivatives of GlcNAc were detected and quantified by
ultraviolet absorption at 210 nm using a previously established
procedure [14], and HP Chemstation software off-line for the pro-
cessing.
2.6. Solubility analyses
The solubility of oNPG, lactose and GlcNAc in water or water/t-
BuOH (70:30, v/v) mixtures was evaluated at 40 ◦C under stirring
conditions by progressively adding into a sample containing the
compound (100 mg of oNPG or 500 mg of lactose or GlcNAc), var-
ious volumes of solvent decreasing within the range 10–1000 L,
the starting volume depending on the compound tested, until com-
plete solubilisation was obtained. The molar concentration of the