42
D. Alagöz et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 40–46
48.5 kDa (fumarase), 58.5 kDa (pyruvate kinase), 90 kDa (lactofer-
2-hydroxy-4-phenylbutyronitrile used as standards for chiral
HPLC analyses were achieved according to Cabirol et al. [22]. The
characterizations of the synthesized cyanohydrins were performed
by using chiral HPLC, FTIR and NMR analyses. 1H NMR and 13C
NMR spectra in DMSO-d6 were recorded on a Bruker Ultrashield
TM NMR 300 MHz spectrometer with tetramethylsilane (TMS) as
the internal standard. Chemical shifts (ı) were expressed in parts
per million (ppm), multiplicity (s, singlet; d, doublet; dd, doublet of
doublets; t, triplet; q, quartet; m, multiplet) and coupling constants
(J) were expressed as Hertz (Hz). A FTIR instrument (Perkin Elmer
Spectrum RX/FTIR system) was used to determine the functional
groups after the pellet was prepared in KBr. HPLC analyses were
performed on Nucleocel Delta S chiral column at 220 nm.
2.2.4. Immobilization of PatHNL
The immobilization procedure was performed according to
Mateo et al. [26]. One gram of each support was treated with 9.0 mL
of the purified PatHNL solution (1.0 mg protein mL−1 prepared in a
phosphate buffer (1.0 M, pH = 7.0)). The immobilization process was
performed at 25 ◦C for 24 h. The immobilized PatHNL preparations
dure was continued until no protein was detected in the filtrates.
The immobilized HNL preparations were stored at 5 ◦C until use.
the filtrates [27].
2.2.7.1. (R/S)-mandelonitrile. 1H NMR ı (ppm): 4.51 (s, 1H, H5C6-
CHOH-CN), 5.51 (s, 1H, H5C6-CHOH-CN), 7.38–7.42 (m, 5H, H5C6-
CHOH-CN).
2.2.5. HNL assay
The lyase activities of HNL preparations were spectrophotomet-
rically determined according to Tükel et al. [23]. Briefly, 2.85 mL of
citrate buffer (50 mM, pH = 5.5) and 0.1 mL of HNL solution were
mixed at 25 ◦C for 2 min. The reaction was initiating by adding 50 L
of (R/S)-mandelonitrile solution (30 mM in absolute ethanol). After
10 min, 0.5 mL of aliquots withdrawn and diluted to 3.0 mL with
acetate buffer (50 mM, pH = 4.0). The amount of formed benzalde-
hyde was measured at 250 nm. The same experiment without HNL
was performed as blank. One unit of HNL activity was defined as the
amount of HNL produced 1.0 mol benzaldehyde in 1 min under
the assay conditions.
13C NMR ı (ppm): 63.2 (H5C6-CHOH-CN), 119.2 (H5C6-CHOH-
CN), 128–135 (H5C6-CHOH-CN).
IR (film) cm−1: 3411, 3064, 2250, 1697, 1494, 1455, 1433, 1284,
1193, 1086, 1041, 1026.
2.2.7.2. (R/S)-2-chloromandelonitrile. 1H NMR ı (ppm): 3.5 (s, 1H,
H4ClC6-CHOH-CN), 5.78 (s, 1H, H4ClC6-CHOH-CN) 7.26–7.65 (m,
4H, H4ClC6-CHOH-CN).
13C NMR ı (ppm): 60.9 (H4ClC6-CHOH-CN), 117.9 (H4ClC6-
CHOH-CN), 128–133 (H4ClC6-CHOH-CN).
IR (film) cm−1: 3395, 3067, 2923, 2250, 2058, 1957, 1921, 1805,
1694, 1593, 1568, 1476, 1443, 1417, 1278, 1245, 1191, 1126, 1056,
1035.
2.2.6. Characterization of PatHNL
2.2.6.1. Lyase activity. The lyase activities of HNL preparations
were measured in 50 mM of acetate buffers (pH = 4.0–5.5), citrate
buffer (pH = 6.0) and phosphate buffers (pH = 6.5 and 7.0). The opti-
mum temperatures of all the HNL preparations were investigated
at different temperatures ranging from 10 to 40 ◦C.
The apparent maximum reaction rate (Vmax) and the apparent
Michaelis–Menten constant (Km) of free and immobilized prepa-
rations were determined by applying the HNL assay for various
mandelonitrile concentrations (0.1–2.0 mM). The activities of free
and immobilized preparations were measured at their optimum
conditions. Enzyme Kinetics Module programme (Sigma-Plot 12.0)
was used for determining the apparent Vmax and the apparent Km
values of HNL preparations.
2.2.7.3. (R/S)-3,4-dihydroxy mandelonitrile. 1H NMR ı (ppm): 3.4
(s, 1H, HOCHOCH3C4-CHOH-CN), 4.35 (s, 1H, HOCHOC C4H3-
CHOH-CN), 4.35 (s, 1H, HOCHOCC4H3-CHOH-CN), 5.2 (s, 1H,
HOCHOCC4H3-CHOH-CN), 7.0 (d, 1H, HOCHOCC4H2H-CHOH-CN),
7.0 (d, 1H, HOCHOCC4H2H-CHOH-CN), 7.1 (s, 1H, HOCHOCC4H2H-
CHOH-CN).
13C NMR
ı (ppm): 60.1 (HOCHOCC4H3-CHOH-CN), 118.2
(HOCHOCC4H3-CHOH-CN), 124.2–128.8 (HOCHOCC4H3-CHOH-
CN), 140.1–140.3 (HOCHOCC4H3-CHOH-CN).
IR (film) cm−1: 3395, 3067, 2923, 2250, 2058, 1957, 1921, 1805,
1694, 1593, 1568, 1476, 1443, 1417, 1278, 1245, 1191, 1126, 1056,
1035.
The storage stabilities of all HNL preparations were investigated
at room temperature and 5 ◦C.
2.2.7.4. (R/S)-2-hydroxy-4-phenylbutyronitrile. 1H NMR ı (ppm):
2.14 (H5C6-CH2-CH2-CHOH-CN), 2.55 (t, H5C6-CH2-CH2-CHOH-
CN), 3.65 (s, H5C6-CH2-CH2-CHOH-CN), 4.22 (t, H5C6-CH2-CH2-
CHOH-CN), 7.27–7.29 (H5C6-CH2-CH2-CHOH-CN).
2.2.6.2. Carboligation activity. The effects of pH and temperature
on the carboligation activities of free and immobilized PatHNL were
tested for (R)-mandelonitrile synthesis. The effect of pH was inves-
tigated in a biphasic TBME–citrate buffer with different pH values
(400 mM, 3.5–6.0). To determine the optimum temperature, the
carboligation activities of free and immobilized HNLs were mea-
sured in the temperature range of 5–25 ◦C in biphasic TBME–citrate
buffer at pH 4.0. The apparent Vmax and the apparent Km values
of free and immobilized PatHNL preparations for the carboliga-
tion activity were determined for the synthesis of mandelonitrile
by using various benzaldehyde concentrations. The carboligation
activities of all the PatHNL preparations in the characterization and
kinetic experiments were measured by using HPLC described in
Section 2.2.8 after 2 h reaction time.
13C NMR ı (ppm): 28.4 (H5C6-CH2-CH2-CHOH-CN), 35.2 (H5C6-
CH2-CH2-CHOH-CN), 63.2 (H5C6-CH2-CH2-CHOH-CN), 118.4
(H5C6-CH2-CH2-CHOH-CN), 126–142 (H5C6-CH2-CH2-CHOH-CN).
IR (film) cm−1: 3338, 3026, 2929, 1773, 1664, 1497, 1453, 1132,
1029.
2.2.8. HNL catalyzed synthesis of (R)-mandelonitrile, (R)-2-chloro
mandelonitrile, (R)-3,4-dihydroxymandelonitrile and
(R)-2-hydroxy-4-phenylbutyronitrile
To 200 L of the free HNL (2 mg mL−1) or 50 mg of immobi-
lized HNL preparations, 500 L of citrate buffer (400 mM, pH = 4.0),
100 L of aldehyde solution (1.0 M in DMSO) and 1.0 mL of TBME
were added at 5 ◦C. The reaction was initiated by adding 200 L of
HCN solution (1.0 M in TBME) prepared according to Bhunya et al.
[28]. Subsequently, 100 L of sample withdrawn from TBME phase
was diluted to 500 L with hexane/2-propanol mixture (96/4, v/v)
and analyzed by a HPLC equipped with Nucleocel Delta S chiral
column. The mobile phase was hexane/2-propanol mixture (96/4,
2.2.7. Chemical synthesis of mandelonitrile,
2-chloromandelonitrile, 3,4-dihydroxy mandelonitrile and
2-hydroxy-4-phenylbutyronitrile
The
chemical
synthesis
of
mandelonitrile,
2-
chloromandelonitrile,
3,4-dihydroxymandelonitrile
and