N. Jangir, et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
conditions for BmHNL biocatalysis.
converts 1 μmol of racemic mandelonitrile to benzaldehyde in one
minute under standard conditions. All the reactions were performed in
triplicates.
In the present study, we investigated the effect of different para-
meters such as pH, temperature, buffer concentrations, presence of
additives/inhibitors, stabilizers, substrate concentrations and organic
solvents on the activity and stability of BmHNL. We also investigated
the effect of organic solvents and temperature over the secondary
structure of BmHNL by circular dichroism (CD) analysis. Our study has
revealed that addition of polyols especially glycerol improves the en-
antioselectivity of BmHNL in the formation of (S)-mandelonitrile up
to > 99% ee compared to 75% ee in the absence of glycerol. Two
previous reports show such increase in the % ee in the BmHNL cata-
lyzed synthesis of (S)-mandelonitrile. They are with (i) engineered en-
zyme, i.e., BmHNL-H103C-N156 G [30] or immobilized BmHNL [29].
Further, sucrose addition has improved BmHNL’s half-life at pH 3.5,
more than five folds as compared to without stabilizer.
2.3. Influence of biophysical parameters on BmHNL stability and activity
We have studied various biophysical parameters to determine the
stability and activity of BmHNL under different conditions. The enzyme
was incubated in different conditions such as different pH, temperature,
buffer concentration, additives, substrate concentrations (as inhibitor),
and stabilizers and their effect on stability and activity of BmHNL were
studied. In the case of each of the above studies, the specific activity of
BmHNL was calculated by using (RS)-mandelonitrile cleavage assay (as
given in 2.2). In case of control reactions, the corresponding buffer was
used instead of the enzyme. All measurements were performed in tri-
plicates.
2. Materials and methods
2.3.1. Effect of pH
BmHNL (LOCUS: AB505969) synthetic gene cloned into pUC57 was
synthesized and procured from Gene Script, USA. Culture media, am-
picillin, and Dialysis membrane bags were purchased from HiMedia
laboratory Pvt Ltd. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was
purchased from BR-BIOCHEM Pvt Ltd. For protein purification, Ni-NTA
agarose superflow resin was purchased from Qiagen. Imidazole, alde-
hydes, mandelonitrile, metal salts and organic solvents used in the
study were purchased from Sigma Aldrich, AVRA, SRL, and Alfa-Aesar.
HPLC grade solvents were obtained from RANKEM, Molychem, FINAR,
and SRL. Different stabilizers, e.g. sorbitol, glycerol, lactose, and su-
crose were purchased from SRL. Acetone cyanohydrin (AcCN) was
purchased from amino organics (local vendor). 10,000 MWCO Amicon
tubes for concentrating protein were obtained from Merck Millipore.
2.3.1.1. On stability. The effect of pH on stability of BmHNL was
studied in 50 mM citrate-phosphate buffer of different pH, e.g. 3.5,
4.0, 4.5, 5.0, 5.5, 6.0, and 6.5. The enzyme found in 20 mM KPB pH 7.0
on purification was subjected to buffer exchange with 50 mM citrate-
phosphate buffer of the corresponding pH, i.e. 3.5–6.5 and was stored
at room temperature. HNL activity of each of the buffer exchanged
enzyme was determined. The activity found was considered as the
initial activity of that enzyme stored at the given pH. Further activities
of BmHNL were measured at different time intervals until the specific
activity reached half of the initial activity.
2.3.1.2. On activity. Specific activity of BmHNL was measured as
described in 2.2, except the assay buffer was replaced by 50 mM
citrate-phosphate buffer of pH 3.5–6.5 in separate experiments
2.1. Expression and protein purification
2.3.2. Effect of temperature
BmHNL enzyme with His-tag was produced in E. coli as per our
previous reports. [23,29]. The crude extract obtained was purified
using Ni-NTA agarose resin. The crude extract was loaded onto a 10 mL
bed-volume of the resin pre-equilibrated with 20 mL of equilibration
buffer (50 mM KPB pH 7.0, 10 mM imidazole, 300 mM NaCl). The resin
was allowed to bind to the enzyme by shaking the column at 4 °C for
45 min, and the unbound protein was collected as flow-through (FT).
The resin was washed twice with 20 mL of wash buffer (50 mM KPB pH
7.0, 20 mM imidazole, 300 mM NaCl) followed by protein eluted with
elution buffer (50 mM KPB pH 7.0, 150 mM imidazole, 300 mM NaCl).
The final elution carried out using a buffer containing a high con-
centration of imidazole (50 mM KPB pH 7.0, 300 mM imidazole,
300 mM NaCl). All steps were carried out at 4 °C. All the fractions were
analyzed by SDS-PAGE (Fig. S1) and protein quantitated by Nanodrop.
Removal of imidazole from eluted protein was done by dialysis by using
pre-treated membrane (20 kDa MWCO). Subsequently, the dialyzed
protein was concentrated using 10’000 MWCO Amicon tubes, and the
enzyme was stored at 4 °C.
2.3.2.1. On stability. Influence of temperature on the stability of
BmHNL was studied by incubating the enzyme in 20 mM KPB pH 7.0
at different temperatures ranging from 10 to 80 °C in a thermoshaker.
The initial activity was measured using HNL assay at room temperature
(as given in 2.2). The activity of enzymes incubated at different
temperatures was measured at multiple time points until each of
them reached half of its corresponding initial activity. A control
experiment was also performed under similar conditions where the
enzyme was replaced by its corresponding buffer.
2.3.2.2. On activity. Specific activity of BmHNL was measured as
described in 2.2, except separate experiments were carried out for
measuring the activity by incubating the reaction mixture in 50 mM
citrate-phosphate buffer pH 5.5, at different temperatures, i.e., 10 to
80 °C in a thermomixer.
2.3.3. Effect of buffer concentrations
2.3.3.1. On stability. The study of the effect of buffer concentrations on
the stability of BmHNL was carried out by storing the purified enzyme
in different concentrations of citrate-phosphate buffer pH 5.5, i.e., 50,
100, 200, 300 and 400 mM. This was performed by an exchange of the
citrate-phosphate buffer of appropriate concentration with the buffer of
the purified enzyme, as described in section 2.3.1. The initial activity
was measured after buffer exchange. The enzyme was stored at room
temperature, and the activity of each enzyme present in different
concentrations of buffer was measured until it reached half of the initial
activity.
2.2. HNL assay via mandelonitrile cleavage
HNL activity was measured by monitoring the formation of ben-
zaldehyde due to cleavage of racemic mandelonitrile, in a microtitre
plate using Multiskan Go UV–vis spectrophotometer (Thermo
Scientific). The reaction mixture contained 175 μL of 50 mM citrate-
phosphate buffer pH 5.0, 20 μL of 70 mM (RS)-mandelonitrile pre-dis-
solved in 5 mM citrate-phosphate buffer pH 3.15. The reaction was
initiated by the addition of 5 μL of BmHNL (0.24 mg/mL), and the rate
of benzaldehyde production was measured at 280 nm over a period of
5 min. A control experiment was carried out in an identical manner
without using enzyme, and the corresponding absorbance was sub-
tracted. One unit is defined as the amount of the enzyme which
2.3.3.2. On activity. The specific activity of BmHNL was measured as
per the protocol described in section 2.2 using citrate-phosphate buffer
pH 5.5 of different concentrations (50, 100, 200, 300 and 400 mM)
instead of only 50 mM citrate-phosphate pH 5.5.
2