Regulation of Enzyme Activity in Au-Doped Reverse Micelles
FULL PAPER
The reverse micelles with GNPs synthesized in situ were also formed by
reduction with only NaBH4 without using trisodium citrate as the capping
agent by following the above protocol.
stock solution (6 mL) was added to the reverse micelle solution (1.5 mL)
in the cuvette, which was shaken to achieve a reaction concentration of
the substrate of 20–250 mm.
2) Enzyme solution: Trypsin stock solution (1.2ꢃ10À3 m) was prepared in
buffer solution (1 mL, pH 3; containing CaCl2 (1 mm)) and diluted ten-
fold with phosphate buffer solution (pH 6, 20 mm) just before use. The di-
luted solution (4 mL) was added to the reverse micelle solution (1.5 mL)
to reach an overall trypsin concentration of 0.32 mm in the cuvette.
Preparation of reverse micelles and GNP-doped reverse micelles: The
requisite quantity of surfactant was dispersed in iso-octane in a 2-mL
volumetric flask to which a calculated amount of n-hexanol was added to
attain the corresponding z value and shaken vigorously. Finally, aqueous
buffer (phosphate) solution was added (to reach the corresponding
W0 value), and the whole suspension was vortexed to obtain a clear ho-
mogeneous solution of CTAB or CTPAB (50 mm)/iso-octane/n-hexanol/
water reverse micelle. A similar protocol was followed to obtain the
GNP-doped reverse micelle. A solution of GNP in water was added in-
stead of buffer. During the preparation of the reverse micelle with a
lower concentration of the GNPs, phosphate buffer solution was added
so that the GNP solution obtained the corresponding W0 value and the
desired GNP concentration.
3) Enzyme kinetics: The kinetic parameters kcat of the compartmentalized
trypsin-catalyzed hydrolysis (trypsin: 0.32 mm) of N-a-benzyloxycarbonyl-
l-lysine-p-nitrophenyl ester hydrochloride in CTAB reverse micelles
were determined spectrophotometrically[11e] by following the formation
of p-nitrophenol at the isosbestic points. The isosbestic point and molar
extinction coefficient at liso of the p-nitrophenol/p-nitrophenolate couple
in CTAB (50 mm)/water/iso-octane/n-hexanol and in GNP-doped CTAB
reverse micelles are shown in Table S1. In a typical experiment, aliquots
of the different substrate stock solutions (6 mL, 20–250 mm in HPLC-
grade DMSO) were added to the reverse micelles (1.5 mL; previously
prepared at the desired surfactant concentration and pH value in a cuv-
ette). The activity of the enzyme was monitored spectrophotometrically
at the isosbestic point by adding the diluted enzyme solution (4 mL) to
the cuvette. The trypsin activity was studied at pH 6 rather than at the
optimum pH value (around 8.0) primarily to prevent spontaneous chemi-
cal hydrolysis of the substrate in the cationic reverse micelles and to fa-
cilitate the operational stability of the enzyme because the formation of
p-nitrophenol is most convenient to follow at steady state. The overall re-
action concentration of trypsin was 0.32 mm. The initial velocities were
plotted against the concentration of the substrate and the experimental
data were fitted to a linearized Michaelis–Menten plot to determine the
kcat values (sÀ1).
Activity of interfacially solubilized lipase in reverse micelles: The
second-order rate constant k2 in the lipase-catalyzed hydrolysis of p-ni-
trophenyl n-octanoate in cationic w/o microemulsions was determined
spectrophotometrically at the isosbestic points (see Table S1 in the Sup-
porting Information) as reported previously.[11a,13d,15,16] In a typical experi-
ment, the aqueous enzyme stock solution (4.5 mL, 0.34 mgmLÀ1) and sub-
strate stock solution in iso-octane (10 mL, 0.45m) were added to the w/o
microemulsion (1.5 mL) previously prepared at the desired surfactant
concentration and pH value (i.e., the pH value of the aqueous buffer sol-
utions used for preparing the w/o microemulsions; the pH value within
the water pool of the w/o microemulsions did not vary significantly:
<1 unit)[16,23] in a cuvette to attain the particular W0 value and reactant
concentrations. Gentle shaking produced clarification of the microemul-
sion within 1 min. The initial linear rate of increase in the absorbance of
liberated p-nitrophenol was recorded at the isosbestic points liso. The
molar extinction coefficients e at isosbestic point and the liso value of the
p-nitrophenol/p-nitrophenolate couple in w/o microemulsions of different
systems with varying GNP concentrations are given in Table S1 in the
Supporting Information. The overall concentrations of lipase and p-nitro-
phenyl n-octanoate were 1.02ꢃ10À6 gmLÀ1 and 3 ꢃ10À3 m, respectively.
Although the lipase was essentially confined to the dispersed water drop-
lets (at the oil/water interface), for simplicity, the concentration of reac-
tants were referred to the overall concentration to avoid the complexity
of the volume fraction of the water droplet in the w/o microemulsions
and the partitioning coefficient of the substrate.[13d,23] Moreover, we mea-
sured the second-order rate constant k2 instead of the first-order Michae-
lis–Menten catalytic constant kcat because the initial rate of the lipase-cat-
alyzed hydrolysis of p-nitrophenyl alkanoate was first order with respect
to the substrate concentration (see Figure S2 in the Supporting Informa-
tion).[13d,15,16,23] The pH aqueous solutions of GNP511, GNP528, GNP529, and
GNP536 were 8.0, 5.6, 5.3, and 5.0, respectively. In the absence of GNPs,
the lipase activities in the control experiments were measured at the re-
spective pH values and all the other experimental conditions were kept
identical. It is already known that lipase activity remains almost same
within the range pH 2–10 in CTAB reverse micelles.[14a] We also observed
that by varying the pH value, the lipase activity remains same as ob-
served for pH 6 (20 mm phosphate buffer).
CD spectra: The CD spectra of lipase and GNP/lipase in reverse micelles
were recorded on Jasco J-815 spectrometer in a 2-mm path-length cell at
l=220–300 nm with a scan speed of 50 nmminÀ1 (in the case of reverse
micelles, the CD spectra could not be measured below l=220 nm due to
an off-scale signal). For GNP/lipase in water, the spectra were recorded
in a 10-mm path-length cell at l=200–300 nm. All the spectra were cor-
rected by subtracting a blank spectrum (without enzyme) and accumulat-
ed six times. The results are expressed in terms of mean residue ellipticity
(degcm2 dmolÀ1). The final concentration of the lipase was kept at
25 mgmLÀ1 in the reverse micelle and 10 mgmLÀ1 in water.
FTIR spectroscopy: For the FTIR spectra of GNP/lipase, lipase solution
(500 mL, 0.34 mgmLÀ1) in water was mixed with GNP528 in water (560 mL,
1.2 mm) and lyophilized for 24 h. For native lipase, the lipase solution
(500 mL, 0.34 mgmLÀ1) was lyophilized. These lyophilized powders were
carefully transferred into a mortar containing IR-grade KBr (ca. 30 mg)
and were ground to prepare the pellet under strictly dry conditions to
prevent absorption of water vapor. This pellet was further dried by stor-
ing in a vacuum desiccator. Spectra of the pellets were recorded and ac-
cumulated 512 times at a resolution of 2 cmÀ1 with intervals of 1 cmÀ1
.
The FTIR spectrum with lyophilized Au (from identical compositions
without lipase) was also recorded and subtracted from the spectrum of
the GNP/lipase mixture to obtain the spectra of just the protein.
Activity of lipase in water+4% EtOH: The second-order rate constant
k2 of the lipase-catalyzed hydrolysis of p-nitrophenyl n-hexanoate in
water was determined spectrophotometrically at the isosbestic points. CV
lipase also obeys second-order kinetics in water+4% EtOH.[16] For total
solubilization of the substrate, we used substrate and enzyme concentra-
tions that were 50-fold lower than that for w/o microemulsions. Thus, the
final concentration of the substrate was 0.06 mm and lipase was 0.02ꢃ
10À6 gcmÀ3. The isosbestic point and molar extinction coefficient at liso
(340 nm) of the p-nitrophenol/p-nitrophenolate couple in water+4%
EtOH without GNPs and with GNPs were 6500 and 6250mÀ1 cmÀ1, re-
spectively.
Quantification of secondary structure of lipase and GNP/lipase: The
amide I region from n˜ =1600 to 1700 cmÀ1 was analyzed to quantify the
secondary structural contents of the enzymes. The different spectra were
smoothed by a nine-point Savitsky–Golay smoothing function with the
help of software supplied with the Perkin–Elmer FTIR spectrometer. A
straight baseline was obtained in the region from n˜ =1750 to 2000 cmÀ1
,
an important prerequisite in obtaining quantitative structural informa-
tion. Fourier deconvolution was performed using the Perkin–Elmer soft-
ware with a line-narrowing factor k=2.3 and half bandwidth of 37 cmÀ1
.
The second-derivative spectrum was obtained from the deconvoluted
spectra with Savitsky–Golay derivative function software with a five-
point window. a-Helical and b-sheet structures were assigned by follow-
ing the previous reports.[19] The relative amounts of the a helices and
b sheets were determined by computing the areas under the assigned
bands. Each sample was measured four times.
Activity measurement of trypsin
1) Substrate solution: A stock solution of the substrate N-a-benzyloxy-
carbonyl-l-lysine-p-nitrophenyl ester hydrochloride (20–250 mm) was
prepared in HPLC-grade dimethyl sulfoxide (DMSO). An aliquot of the
Chem. Eur. J. 2010, 16, 1941 – 1950
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1949