Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization and Application in Synthesis of Enantiopure Mandelonitrile

Article abstract of DOI:10.1007/s10562-016-1725-6

Abstract: Hydroxynitrile lyases (HNLs) are increasingly finding application in synthesis of enantiomerically pure cyanohydrins. Cyanohydrins are important intermediates in the production of pharmaceuticals and agrochemicals. In the present studies seeds of wild apricot (Prunus armeniaca L.) have emerged as potential source of hydroxynitrile lyase. The HNL of wild apricot (ParsHNL) was purified 8.1 fold and 18.2?% yield with a specific activity of 141 units?mg^?1 protein. The SDS-PAGE of the enzyme revealed that it consists of subunits of 40 and 37?kDa. However, the molecular weight of holoenzyme was assessed to be 360?kDa. The enzyme showed maximum activity in 0.1?M sodium-citrate buffer having pH 4.75 at 25?°C. Thermostability studies revealed that this HNL showed activity up to 70?°C temperature and quite stable up to 50?°C. Activation energy of ParsHNL was calculated to be 37.83?kJ?mol^?1. This enzyme has K_m of 3.76?mM, V_max of 188.4?μmol?mg^?1 min^?1 and k_cat of 1130.4?s^?1 using mandelonitrile as substrate while for reverse reaction using benzaldehyde as substrate it showed K_m of 16.1?mM, V_max of 7.21?μmol?mg^?1 min^?1 and k_cat of 43.3?s^?1. Synthesis of mandelonitrile was carried out using ParsHNL and finally 8.88?mmole (1.184?g) of mandelonitrile was recovered which corresponded to 89?% molar conversion with 96?% ee for R-mandelonitrile. The yield of mandelonitrile was 411?μmol?mg^?1h^?1. These results indicated that ParsHNL has very high potential for synthesis of cyanohydrins and can be used for the production of enantiopure cyanohydrins. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-016-1725-6

Catal Lett (2016) 146:1118–1127
DOI 10.1007/s10562-016-1725-6
Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.):
Purification, Characterization and Application in Synthesis
of Enantiopure Mandelonitrile
Mohammad Asif1 Tek Chand Bhalla1
•
Received: 16 January 2016 / Accepted: 1 March 2016 / Published online: 2 April 2016
Ó Springer Science+Business Media New York 2016
Abstract Hydroxynitrile lyases (HNLs) are increasingly
finding application in synthesis of enantiomerically pure
cyanohydrins. Cyanohydrins are important intermediates in
the production of pharmaceuticals and agrochemicals. In the
present studies seeds of wild apricot (Prunus armeniaca L.)
have emerged as potential source of hydroxynitrile lyase.
The HNL of wild apricot (ParsHNL) was purified 8.1 fold
and 18.2 % yield with a specific activity of 141 units mg-1
protein. The SDS-PAGE of the enzyme revealed that it
consists of subunits of 40 and 37 kDa. However, the
molecular weight of holoenzyme was assessed to be
360 kDa. The enzyme showed maximum activity in 0.1 M
sodium-citrate buffer having pH 4.75 at 25 °C. Ther-
mostability studies revealed that this HNL showed activity
up to 70 °C temperature and quite stable up to 50 °C.
Activation energy of ParsHNL was calculated to be
37.83 kJ mol-1. This enzyme has Km of 3.76 mM, Vmax of
188.4 lmol mg-1 min-1 and kcat of 1130.4 s-1 using
mandelonitrile as substrate while for reverse reaction using
benzaldehyde as substrate it showed Km of 16.1 mM, Vmax
of 7.21 lmol mg-1 min-1 and kcat of 43.3 s-1. Synthesis of
mandelonitrile was carried out using ParsHNL and finally
8.88 mmole (1.184 g) of mandelonitrile was recovered
which corresponded to 89 % molar conversion with 96 % ee
for R-mandelonitrile. The yield of mandelonitrile was
411 lmol mg-1h-1. These results indicated that ParsHNL
has very high potential for synthesis of cyanohydrins and
can be used for the production of enantiopure cyanohydrins.
& Tek Chand Bhalla
bhallatc@rediffmail.com
1
Department of Biotechnology, Himachal Pradesh University,
Summer Hill, Shimla, Himachal Pradesh 171005, India
123

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization…
1119
Graphical Abstract
Crude HNL
Seed of Wild Apricot
Meal
Purification of HNL
Synthesis of Cyanohydrin
Hanes-Woolf Plot
Activation
Energy
Thermostability & Half Life
123

1120
M. Asif, T. C. Bhalla
2.2 Preparation of Crude Enzyme Extract
Keywords Cyanohydrin Á ParsHNL Á Wild apricot Á
Mandelonitrile Á Hydroxynitrile lyase Á Prunus armeniaca
Crude hydroxynitrile lyase was extracted from the kernel of P.
armeniaca seeds according to method outlined by Han et al. [19]
with slight modification. In brief, the seeds were soaked in dis-
tilled water for 24 h at room temperature, washed with distilled
water and ground with chilled ethyl acetate. It was air dried and
stored at 4 °C and termed as meal. The crude HNL was prepared
by suspending this meal in distilled water (7.4 g/100 mL), its pH
adjusted to 7.4 with 1 N NH4OH (for effective extraction of
enzyme) and was kept at 4 °C overnight. It was centrifuged at
15,0009g for 15 min and supernatant was collected and its pH
was adjusted to 5.5 with 50 % acetic acid. It was filtered and the
filtrate was used as crude enzyme in subsequent experiments.
The protein concentration in the enzyme preparation was
determined following the method of Bradford [20].
1 Introduction
Hydroxynitrile lyases (HNLs, EC 4.1.2.x) are the enzymes
which mediate the release of hydrogen cyanide (HCN) and
aldehyde or ketone from cyanohydrins [1] and catalyse
enantioselective synthesis of cyanohydrins [2]. The
cyanohydrins and their derivatives are finding wide appli-
cations in industries for production of pharmaceuticals,
agrochemicals and cosmetics, since these can be readily
converted into other synthetically relevant building blocks
like a-hydroxy carboxylic acids, a-hydroxy ketones and b-
amino acids [3].
The HNLs have been reported, purified and character-
ized from several plants e.g. almond (Prunus amygdalus),
flax (Linum usitatissimum), cherry (Prunus serotina),
peach (Prunus persica), capulin (Prunus capuli), rosary
pea (Abrus precatorius), rubber tree (Hevea brasiliensis),
cherrylaurel (Prunus laurocerasus), cassava (Manihot
esculenta), Japanese apricot (Prunus mume), Prunus lyonii,
Phlebodium aureum, Ximenia americana, Eriobotrya
japonica, Passiflora edulis, etc. [1, 4–17].
2.3 Assay of Hydroxynitrile Lyase Activity
The activity of HNL was assayed according to the method of
Siritunga et al. [21] in 1 ml reaction mixture comprising 0.1 M
sodium citrate buffer (pH 5.0) containing 10 lmoles of man-
delonitrile and 5 ll crude enzyme (3 lg protein), at 25 °C in
water bath for 30 min, against a control reaction (without
enzyme). The liberated cyanide was estimated colourimetri-
cally at 585 nm wavelength and one unit of enzyme activity was
defined as lmole of cyanide formed min-1 mg-1 of protein.
In the present investigation, purification and character-
ization of HNL from seeds of wild apricot (Prunus arme-
niaca L.) (ParsHNL) was undertaken because the seeds are
by-product of apricot processing industry. Use of these
seeds as a source of enzyme is an economically and eco-
logically interesting alternative to their existing processing
and utilization. The seeds of P. armeniaca have been
shown to contain hydroxynitrile lyase and b-glucosidase,
but have not been explored so far. Though a crude HNL
preparation from a cultivated variety of P. armeniaca i.e.
white apricot (shakarpara) (which is more juicy but less
acidic than wild apricot) has been used for synthesis of
several cyanohydrins [18], but no study has been carried
out on purification and characterization of HNL of P.
armeniaca. Therefore, in the present studies an attempt has
been made to purify and characterize ParsHNL and to use it
for the synthesis of mandelonitrile.
2.4 Purification of ParsHNL
All steps of protein purification were performed at 4 °C
using 0.1 M sodium citrate buffer (pH 5.0) and centrifu-
gation was carried out at 15,0009g for 15 min at 4 °C. The
crude enzyme was prepared from 1.42 g meal of wild
apricot following the procedure described in Sect. 2.2.
2.4.1 Ammonium Sulphate Precipitation
Ammonium sulphate fractionation of crude enzyme
preparation (12 mg crude enzyme protein in 20 ml buffer)
was done to concentrate HNL activity.
2.4.2 Gel Permeation Chromatography
The gel permeation chromatography was carried out using
Sephacryl S-100 column. Dialysed sample was applied on
to the pre-equilibrated column with start buffer (0.1 M
sodium citrate buffer pH 5.0), eluted with elution buffer
(0.1 M sodium citrate buffer pH 5.0 containing 0.1 M
NaCl) with flow rate of 30 ml/h and fractions of 3 ml were
collected. The fractions rich in HNL activity were pooled,
analysed and termed as GPC HNL preparation.
2 Materials and Methods
2.1 Plant Material
Seeds of wild apricot (P. armeniaca) used as source of
HNL were obtained from the Department of Food Science
and Technology, Dr. Y S Parmar University of Horticulture
and Forestry, Solan, India.
123

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization…
1121
2.4.3 Ion Exchange Chromatography
Laemmli [22] (Fig. 1) . The molecular mass of the native
enzyme was determined by gel-filtration chromatography
on a Sephacryl S-300 column HR 16/60 (GE Healthcare)
by comparing the elusion volume of reference proteins
markers (Fig. 2).
The ion exchange chromatography was performed with
DEAE-Sepharose anion exchanger column. The GPC HNL
preparation was applied on to the pre-equilibrated column,
which was eluted with start buffer (0.1 M sodium citrate
buffer pH 5.0). Fractions of 1 ml each were collected with
a flow rate of 60 ml/h and the protein fractions rich in HNL
activity were pooled and analysed electrophoretically. The
enzyme preparation at this stage was designated as IEC.
2.4.5 Determination of FAD of ParsHNL
The purified enzyme was denatured by heating in boiling
water bath for 10 min and FAD was estimated after fil-
teration by HPLC in supernatant using 70 % acetonitrile in
water as mobile phase using 515 HPLC pump (Waters)
equipped with SunFire C18 5 lm (4.6 9 250 mm) column
(Waters) and 2998 Photodiode Array Detector (Waters)
with flow rate of 60 ml/h. The absorption was taken at
263 nm.
2.4.4 Sodium Dodicyl Sulphate Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
The enzyme preparations of various stages of purification
were subjected to SDS-PAGE using the procedure of
2.5 Optimization of Reaction Conditions for Assay
of ParsHNL
Various parameters were optimized to determine ideal
reaction conditions for assay of ParsHNL activity. The
effect of substrate concentration on ParsHNL activity was
also studied using mandelonitrile as substrate. The activity
of the purified ParsHNL was determined at different pH
(3.5–6.5) using several buffer systems [sodium citrate,
sodium acetate, sodium phosphate, potassium phosphate
and citrate phosphate] of molarity 0.1 M using mande-
lonitrile as substrate. Effect of temperature (ranging from
20 to 50 °C) was studied on the activity of purified
ParsHNL, to determine optimum reaction temperature. The
activation energy of the enzyme was also determined.
Effect of various metal ions (at 10 mM conc.) on ParsHNL
activity was also evaluated (Table 2).
Fig. 1 SDS-PAGE of purified ParsHNL. Lane 1 was loaded with
molecular mass standard: ovalbumin (43 kDa), carbonic anhydrase
(29 kDa), soyabeen trypsin inhibitor (20 kDa), lysozyme (14.3 kDa),
aprotinin (6.5 kDa) and insulin chain b (3.5 kDa). Crude enzyme
(lane 2–4) and purified ParsHNL (lane 5)
800
720
700
600
500
480
400
360
300
242
200
146
100
66
0
35
40
45
50
55
60
65
70
Elusion volume (ml)
Fig. 2 Non-denaturating PAGE of purified ParsHNL and determina-
tion of molecular mass. Lane 1 was loaded with molecular mass
standard: apoferritin band 1 (720 kDa), apoferritin band 2 (480 kDa),
b-phycoerythrin (242 kDa), lactate dehydrogenase (146 kDa), bovine
serum albumin (66 kDa) and soybean trypsin inhibitor (20 kDa).
Crude enzyme (lane 2–3), gel permeation chromatography (lane 4)
and purified ParsHNL (lane 5)
123

1122
M. Asif, T. C. Bhalla
2.6 Thermostability and Determination of Half-Life
of the Enzyme
515 HPLC pump (Waters) equipped with SunFire C18 5 lm
(4.6 X 250 mm) column (Waters) and 2998 Photodiode
Array Detector (Waters) with flow rate of 60 ml/h. To
determine the enantiomeric excess (ee %), chiral chro-
matography was performed using Chiralcel-OJH 5 lm
(4.6 9 250 mm) column (Daicel) and SPD-10A vp UV
detector, with mobile phase of n-hexane/isopropanol in ratio
of 80/20 respectively with flow rate of 60 ml/h. Absorbance
was recorded at 210 nm and data analysed using Shimadzo
LC solution software. The % ee was calculated as
For determination of half-life of the enzyme and its ther-
mostability, it was pre-incubated at different temperature
(25, 30, 50, 60 and 70 °C) up to 72 h and its activity was
determined at regular interval of time. In order to assess the
denaturation of ParsHNL, its activity reduction with time
was expressed as:
d½EŠ
RÀS
¼ Àkd  ½EŠ
ð1Þ
 100. A blank reaction was also performed without
RþS
dt
enzyme and the amount of mandelonitrile formed in blank
was substracted from enzyme catalysed reaction.
where [E] denotes HNL activity at time t min; kd the first-
order enzyme deactivation constants of HNL s-1. Inte-
grating Eq. 1, we obtain:
½EŠ ¼ ½EoŠeÀkdt
where [Eo] represents original HNL activity at t = 0.
ð2Þ
3 Results and Discussion
3.1 Extraction of Crude Enzyme
Solving for ln ([Eo]/[E]) from Eq. 2, we obtain:
ꢀ
ꢁ
12 mg crude HNL protein with 209 units (specific activity
17.4 U/mg protein) could be prepared from 1.42 g meal.
Earlier Pratush et al. [8] were able to extract 24.32 mg of
crude HNL protein (64 U/mg) from 11.1 g meal of rosary
pea while Techawaree et al. [16] extracted 1000 mg of crude
HNL protein using 2 kg seeds of Eriobotrya japonica with
specific activity of 0.8 U/mg of protein. The recovery of
protein was found to be 0.84, 0.22 and 0.05 (% mg protein
obtained from per mg meal/seed used) for wild apricot,
rosary pea and Eriobotrya japonica respectively. It reflects
either the procedure followed was efficient or the material
used i.e. seed of wild apricot are rich in HNL.
Eo
E
ln
¼ kd  t
ð3Þ
From Eq. 3, it has been found that ln ([Eo]/[E]) changes
linearly with thermal duration t with a slope of kd. The half-
life of ParsHNL (t1/2), was derived from Eq. 4 as
following:
0:693
t1=2
¼
ð4Þ
kd
2.7 Determination of Km and Vmax of ParsHNL
Hyperbolically fitted plots drawn between V and [S] were
used to determine various kinetic parameters (Km, Vmax
cat, kcat/Km and Km/Vmax) of purified ParsHNL using
3.2 Purification of ParsHNL
,
k
The purification of ParsHNL was carried out by using
ammonium sulphate precipitation, gel filtration and ion
exchange chromatographies. The maximum increase in
specific activity (45 U/mg protein) was observed in
30–40 % ammonium sulphate saturation with a yield of
50.8 % and 2.6 fold of purification (Table 1). The gel
permeation chromatography was done and the fraction
number 9 and 10 showed high HNL activity, showing 4.9
fold purification with 31.3 % yield of ParsHNL. The
DEAE Sepharose ion exchange chromatography fractions
numbering 2 to 6 having high HNL activity were pooled
and this exhibited 8.1 fold purification with a yield of
18.2 % of the enzyme (Table 1). Previously, 4.3 fold
purification with 60 % yield in Prunus lyonii [13], 122 fold
purification with 38 % yield in Ximenia americana [15],
1.71 fold purification with 50 % yield in Linum usitatissi-
mum [4, 24], 49 fold purification with 36 % yield in Eri-
obotrya japonica [16] and 9 fold purification with 26 %
yield in rosary pea [8] had been reported. Most of the HNL
mandelonitrile as substrate (1–20 mM, 25 °C) for catalysis
of mandelonitrile into benzaldehyde and HCN, as well as
for determining the kinetic parameters for synthesis reac-
tion of ParsHNL using benzaldehyde as substrate
(4–16 mM).
2.8 Synthesis of Mandelonitrile Using ParsHNL
The procedure of Zandbergen et al. [23] was used for syn-
thesis of mandelonitrile using purified ParsHNL with slight
modification. In a flat bottom flask, 650 mg of KCN (in ethyl
acetate) and 1.016 ml benzaldehyde (10 mmole; in DMSO)
with 1.35 mg (190.3 units) of ParsHNL were added to it and
final volume was made to 100 ml with ethyl acetate. The
reaction was continued while stirring for 16 h at 4 °C.
Mandelonitrile synthesized in the reaction was quantified by
HPLC using 65 % acetonitrile in water as mobile phase using
123

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization…
Table 1 Purification of ParsHNL
1123
Stage of purification
Total protein (mg)
Specific activity (U/mg protein)
Total activity (units)
Yield (%)
Purification (fold)
Crude enzyme
ASF
12
17.4
45
209
100
1
2.36
0.77
0.27
106.2
65.4
38.1
50.8
31.3
18.2
2.6
4.9
8.1
GPC
85
IEC
141
Specific activity = One unit of enzyme activity was defined as lmole of cyanide formed min-1 mg-1 of protein
ASF Ammonium sulphate fraction; IEC Ion exchange chromatography; GPC Gel permeation chromatography
Fig. 3 Effect of buffer system
on ParsHNL activity. The
activity of HNL was estimated
with various buffers system
with salinity of 0.1 M
160
140
120
100
80
60
40
20
0
Sodium citrate
Sodium acetate
Potassium
phosphate
Sodium
phosphate
Citrate
phosphate
Buffer system
of Rosaceace family contains FAD as co-factor, hence
possibilities of the presence of FAD as cofactor of
ParsHNL was investigated. It was observed that ParsHNL
is FAD containing enzyme, as clear peak of FAD was
observed in supernatant corresponding to FAD standard.
From the enzyme solution (3.2 mg ml-1) the observed
FAD concentration was found to be 77.6 mM in super-
natant after denaturation.
(75.2 U/mg protein), sodium phosphate (94.1 U/mg pro-
tein), potassium phosphate (71.9 U/mg protein) and citrate
phosphate (15.5 U/mg protein) buffers (each having
salinity of 0.1 M) was estimated and sodium citrate buffer
of molarity 0.1 M turned out to be best for HNL activity
(Fig. 3). Effect of substrate (mandelonitrile) concentration
was studied and it was observed that ParsHNL showed
highest activity at 12 mM reaction mixture (Fig. 7).
ParsHNL showed optimum activity at pH 4.75–5.0 with
mandelonitrile (Fig. 4) in 0.1 M sodium citrate buffer.
Among the metal ions tested, mercuric ion (Hg2?) com-
pletely inhibited the activity of ParsHNL. This suggest, that
the ParsHNL has cysteine residue at its active site which is
also supported by the crystal structure of HNL from P.
The electrophoretic analysis of the purified ParsHNL
revealed that its molecular mass was approximately
360 kDa (as estimated by gel permeation chromatography
as well as Native-PAGE; Fig. 2) consisting of heteromeric
units having size of 40 and 37 kDa subunits (as revealed by
SDS-PAGE; Fig. 1). Earlier Siriporn and Montri [25] have
reported homotetramer of HNL with subunit molecular
weight of 25.6 kDa in cassava while HNL of Eriobotrya
japonica has a molecular size of 72 kDa [16]. Pratush et al.
[8] have purified HNL of rosary pea having a molecular
mass of 205 kDa comprising of two types of subunits with
molecular mass of 42 and 36.5 kDa.
dulcis (1ju2) [26]. Other metal ions i.e. Sr2?, Cs2?, Co2?
,
Cu2?, Fe2?, Fe3?, Mn2? and Pb2? showed partial inhibi-
tion of HNL activity and the rest had either very less or no
effect on activity of ParsHNL (Table 2).
3.4 Effect of Temperature on ParsHNL Activity
3.3 Characterization of purified ParsHNL
HNL activity was assayed at different temperature range
(20–50 °C) and 25 °C turned out to be an optimum tem-
perature for the assay of this enzyme (Fig. 5). The increase
in incubation temperature beyond 30 °C resulted in sig-
nificant loss in the enzyme activity. Initial rate of reaction
at different temperatures plotted in Arrhenius format [Ln
The different reaction conditions like buffer system and pH
were optimized for the purified ParsHNL. In order to select
a suitable buffer system for assay of ParsHNL, its activity
in sodium citrate (139 U/mg protein), sodium acetate
123

1124
M. Asif, T. C. Bhalla
Fig. 4 Effect of pH on
ParsHNL activity. The activity
of HNL was estimated at
various pH ranging from 3.5 to
6.5
200
180
160
140
120
100
80
60
40
20
0
3.5
4
4.5
4.75
5
5.25
5.5
6
6.5
pH
transcyanation of acetyltrimethylsilane (ATMS) and its
carbon analogue 3,3-dimethyl-2-butanone (DMBO) with
acetone cyanohydrins and found activation energy of 37.0
and 49.0 kJ/mol for ATMS and DMBO respectively.
Table 2 Effect of various metal ions on ParsHNL
Metal ion
% Residual activity
Control
Strontium (Sr?2
100
)
84.6
89.3
95.3
78.2
78.9
90.0
70.7
77.8
83.0
96.6
98.0
99.0
62.7
0.0
Cesium (Cs?2
Tungsten (W?6
Cobalt (Co?2
Copper (Cu?2
Magnesium (Mg?2
Ferrous (Fe?2
Ferric (Fe?3
Manganese (Mn?2
Cadmium (Cd?2
Zinc (Zn?2
Calcium (Ca?2
Lead (Pb?2
Mercury (Hg?2
)
3.5 Thermostability and Determination of the Half-
Life of the ParsHNL
)
)
Based on the first-order enzyme deactivation assumption
)
and Eq. 3, the ParsHNL deactivation rate coefficient kd at
)
25, 30, 50, 60 and 70 °C are 0.89 9 10-6, 1.36 9 10-6
,
)
2.38 9 10-5, 7.67 9 10-5 and 25.19 9 10-5 s-1 respec-
tively (Table 3), and their corresponding half-life (t1/2) are
216, 141, 8.1, 2.5 and 0.77 h (Fig. 6). ParsHNL does not
show any significant loss of activity at 4 °C, hence it has
good potential for industrial application.
)
)
)
)
)
)
3.6 Km and Vmax of ParsHNL
)
(k) vs. 1/T] yielded a linear profile from which activation
energy of 37.83 kJ/mol was calculated which showed
high catalytic efficiency of the enzyme. Earlier Xu et al.
[27] reported activation energy of MeHNL-catalyzed
Characterization of an enzyme for its kenetic parameters is
important in view of its industrial applicability. ParsHNL
showed Vmax of 188.4 lmol min-1mg-1 protein, Km of
3.76 mM, kcat of 1130.4 s-1, kcat/Km of 300.6 mM-1 -1
s
Fig. 5 Effect of temperature on
ParsHNL activity. The activity
of HNL was determined at
various temperatures ranging
from 20 to 50 °C
200
180
160
140
120
100
80
60
40
20
0
20
25
30
40
50
Temperature (ºC)
123

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization…
1125
and Vmax of HNL reported from different sources i.e. Ph-
lebodium aureum [14], Prunus lyonii [13] and Linum usi-
tatissum [4, 24] were 0.83 mM benzaldehyde and
60.1 lmol min-1 mg-1 protein, 93 mM mandelonitrile and
450 lmol min-1 mg-1 of protein, and 2.5 mM benzalde-
hyde and 1.11 lmol min-1 mg-1 of protein respectively.
These kinetic parameters exhibited that ParsHNL has good
affinity for substrate and is among one of the very active
HNLs reported till date.
Table 3 Half-life and deactivation constant (kd) of ParsHNL
Sl. No.
Temperature (°C)
kd (s-1
)
t1/2 (h)
1
2
3
4
5
70
60
50
30
25
25.19 9 10-5
7.67 9 10-5
2.38 9 10-5
1.36 9 10-6
0.89 9 10-6
0.77
2.5
8.1
141
216
and Km/Vmax of 0.02 mM lmol-1min mg with mande-
lonitrile as substrate (Fig. 7; Table 4). The observed values
of kinetic parameters with benzaldehyde as substrate were
Vmax of 7.21 lmol min-1mg-1 protein, Km of 16.07 mM,
3.7 Synthesis of Mandelonitrile Using ParsHNL
The potential of this enzyme to synthesize mandelonitrile
(cyanohydrins) in reverse reaction was explored. In the
reaction mixture 10 mmole (1.016 ml) of benzaldehyde
kcat of 43.3 s-1, kcat/Km of 2.7 mM-1 -1
and Km/Vmax of
s
2.23 mM lmol-1 min mg (Fig. 8; Table 4), whereas Km
Fig. 6 Determination of
Thermostability of HNL
2.2
2
deactivation rate coefficient (kd)
and half-life (t1/2) of ParsHNL.
The ln of (EO/E) was plotted
against incubation time of
70 C
60 C
50 C
1.8
1.6
1.4
1.2
1
enzyme at various temperatures
0.8
0.6
0.4
0.2
0
0
30
60
90
120
150
180
210
240
270
Time (Min.)
Fig. 7 Hyperbolically fit V vs.
[S] plot. Kinetic parameters of
purified ParsHNL were
determined using mandelonitrile
as substrate
123

1126
M. Asif, T. C. Bhalla
Reference
Table 4 Kinetic properties of ParsHNL and some other HNLs
Sl. no
Substrate
Vmax
Km
kcat
kcat/Km
Km/Vmax
Source
1
2
3
4
5
6
7
8
9
Benzaldehyde
Mandelonitrile
Benzaldehyde
Mandelonitrile
Benzaldehyde
Mandelonitrile
Mandelonitrile
Benzaldehyde
Mandelonitrile
7.21
188.4
60.1
450
16.1
3.76
0.83
93
43.3
1130.4
–
2.7
300.6
–
2.23
P. armeniaca
Present study
Present study
[14]
0.02
P. armeniaca
0.014
0.207
2.25
Phlebodium aureum
P. lyonii
–
–
[13]
1.11
625
2.5
–
–
Linum usitatissum
Abrus precatorious
[4, 24]
[8]
13
2135.4
328.3
4.5
164.3
864
0.32
348
0.021
0.002
5.18
197
0.38
14
P. amygdalus turcomanica Lincz.
P. amygdalus turcomanica Lincz.
Eriobotrya japonica
[29]
[29]
[16]
2.7
46.5
0.161
56
0.003
Fig. 8 Hyperbolically fit V vs.
[S] plot. Reverse kinetic
parameters of purified ParsHNL
were determined for synthesis
of mandelonitrile using
benzaldehyde as substrate
and 10 mmole of potassium cyanide were used in 100 ml
reaction mixture and finally 8.88 mmole (1.184 g) of
mandelonitrile was recovered which corresponded to 89 %
molar conversion. The yield of mandelonitrile was
411 lmol mg-1 h-1. The ParsHNL synthesizes (R)-man-
delonitrile preferencialy as observed in chiral HPLC with
96 % ee. The percentage molar conversion rate was much
higher as compared to previously reported HNLs e.g. 73 %
in case of HNL of Prunus capuli and 61 % molar con-
version by HNL of Mammca americana [7] and was
comparable to HNL of Abrus precatorius i.e. 90 % [8],
almond (Prunus amygdalus; 99 % ee and 100 % conver-
sion), plum (P. domestica; 99 % ee and 92 % conversion)
and peach (P. persica; 99 % ee and 94 % conversion) [28].
beneficial for the pharmaceutical, agrochemicals and cos-
metics industries due to its cost effectiveness. The stability
studies revealed that ParsHNL could be succesfully used
for the industrial uses without any significant loss of
activity at its operational temperature. The results of vari-
ous biochemical and kinetic properties (Km and Vmax
)
alongwith synthesis of mandelonitrile indicated that
ParsHNL is (R)-selective HNL and has high potential for
enantiopure synthesis of R-cyanohydrins.
Acknowledgments Authors are highly grateful to Department of
Biotechnology (DBT), Ministry of Science and Technology, Govt. of
India and University Grants Commission (UGC), New Delhi, India
for providing financial assistance to Mr. Mohammad Asif. The
authors also acknowledge Dr. U. C. Banerjee, Professor & Head,
Pharmaceutical Technology, In-charge Biotechnology, National
Institute of Pharmaceutical Education and Research, SAS Nagar,
Punjab, India, for Chiral analysis of cyanohydrin synthesized.
4 Conclusion
Present study revealed that wild apricot (P. armeniaca) is a
good source of HNL for enantioselective synthesis of
cyanohydrins. Wild apricot contains much more amount of
HNL as compared to other sources and thus it could be
References
1. Sharma M, Sharma NN, Bhalla TC (2005) Enzyme Microb
Technol 37:279
123

Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization…
1127
2. Conn EE (1980) Ann Rev Plant Physiol 31:433
3. Gregory RJH (1999) Chem Rev 99:3649
17. Techawaree U, Tamura K, Ohmiya T, Kittikun AH, Asano Y
(2010) Enzyme Microb Technol 46:456
4. Xu LL, Singh BK, Conn EE (1988) Arch Biochem Biophys
263:256
18. Bhunya R, Mahapatra T, Nanda S (2009) Tetrahedron Asym
20:1526
5. Yemm RS, Poulton JE (1986) Arch Biochem Biophys 247:440
6. Asano Y, Tamura K, Doi N, Ueatrongchit T, Kittikun AH,
Ohmiya T (2005) Biosci Biotechnol Biochem 69:2349
19. Han S, Chen P, Lin G, Huang H, Li Z (2001) Tetrahedron Asym
12:843
20. Bradford MM (1976) Anal Biochem 72:248
21. Siritunga D, Arias-Garzon D, White W, Sayre RT (2004) Plant
Biotechnol J 2:37
22. Laemmli UK (1970) Nature 227:680
23. Zandbergen P, van der Linden J, Brussee J, van der Gen A (1991)
Synth Commun 21:1387
24. Albrecht J, Jansen I, Kula MR (1993) Biotechnol Appl Biochem
17:191
´
´
7. Solıs A, Luna H, Perez HI, Manjarrez N, Sanchez R, Marta AV,
Castillo R (1998) Biotechnol Lett 20:1183
8. Pratush A, Sharma M, Seth A, Bhalla TC (2011) J Biochem
Technol 3:274
9. Wajant H, Forster S (1996) Plant Sci 115:25
10. Gerstner E, Kiel U (1975) Hoppe-Seyler’s Z Physiol Chem
356:1853
11. Huges J, Carvalho FJ, Huges MA (1994) Arch Biochem Biophys
311:496
12. Nanda S, Kato Y, Asano Y (2005) Tetrahedron 61:10908
13. Xu LL, Singh BK, Conn EE (1986) Arch Biochem Biophys
250:322
25. Siriporn C, Montri C (1996) Arch Biochem Biophys 334:401
26. Dreveny I, Gruber K, Glieder A, Thompson A, Kratky C (2001)
Structure 9:803
27. Xu R, Zong MH, Liu YY, He J, Zhang YY, Lou WY (2004) Appl
Microbiol Biotechnol 66:27
¨
14. Wajant H, Forster S, Selmar D, Effenberger F, Pfizenmaier K
(1995) Plant Physiol 109:1231
28. Solic A, Solic-Oba M, Perez HI, Manjarrez N, Cassani J (2011)
Biosci Biotechnol Biochem 75:985
¨
¨
29. Alagoz D, Tukel SS, Yildirim D (2014) J Mol Catal: 101:40
15. Gary WK, Conn EE (1989) Proc Natl Acad Sci USA 86:6978
16. Techawaree U, Kayo A, Komeda H, Asano Y, Kittikun AH
(2008) Biosci Biotechnol Biochem 72:1513
123