Partial purification and immobilization of a new (R)-hydroxynitrile lyase from seeds of Prunus pseudoarmeniaca

Article abstract of DOI:10.1016/j.molcatb.2010.05.001

Hydroxynitrile lyase (HNL) from seeds of Prunus pseudoarmeniaca was partially purified by (NH₄)₂SO₄ fractionation and covalently immobilized onto Eupergit C and Eupergit C 250 L. The percentages of bound protein per gram o

Full text of DOI:10.1016/j.molcatb.2010.05.001

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 161–165
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Partial purification and immobilization of a new (R)-hydroxynitrile lyase from
seeds of Prunus pseudoarmeniaca
∗
S. Seyhan Tükel, Deniz Yildirim , Dilek Alagöz, Özlem Alptekin, Güzide Yücebilgi c¸ , Ramazan Bilgin
University of Cukurova, Faculty of Arts and Sciences, Department of Chemistry, 01330 Adana, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxynitrile lyase (HNL) from seeds of Prunus pseudoarmeniaca was partially purified by (NH ) SO
4
4
2
Received 18 January 2010
Received in revised form 30 April 2010
Accepted 6 May 2010
fractionation and covalently immobilized onto Eupergit C and Eupergit C 250 L. The percentages of bound
protein per gram of Eupergit C and Eupergit C 250 L were about 81 and 98 of the initial amount of protein,
respectively. Km and Vmax values were determined 2.23 mM and 0.54 U/mg prot. for the free HNL, 1.60 mM
and 0.87 U/mg prot. for the immobilized HNL onto Eupergit C and 1.03 mM and 0.35 U/mg prot. for the
immobilized HNL onto Eupergit C 250 L, respectively at optimized reaction conditions. The half lives
Available online 13 May 2010
Keywords:
(
2
t1/2) and the thermal inactivation rate constants (ki) of free and immobilized HNLs were determined at
5 and 50 C, immobilized HNLs displayed higher thermal stability. Carboligation activities of free and
Hydroxynitrile lyase
Immobilization
Eupergit
◦
immobilized HNLs for (R)-mandelonitrile (R-MN) synthesis were also determined. Besides, reusabilities
of immobilized HNLs for both lyase and carboligation activities were investigated by using batch type
reactors.
(
R)-Mandelonitrile
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
under mild conditions and generate less waste than conventional
organic synthesis. However, lack of the operational stability and
high price of enzymes are main problem in commercial production.
On the other hand, enzyme immobilization techniques generally
offer several advantages for industrial and biotechnological appli-
cations, including repeated use, ease of separation of reaction
products from the biocatalyst, improvement of enzyme stability,
continuous operation in a packed-bed reactor and the alteration of
the properties of the enzyme.
Eupergit supports were used by many researchers as carriers
for immobilization of various enzymes. It was reported that these
carriers were very stable and had good chemical and mechan-
ical properties (simple immobilization procedure, high binding
capacity, low water uptake, high flow rate in column proce-
dures, excellent performance in stirred bath reactors, etc.) [2–4].
Eupergits were made by copolymerization of N,N-methylene-
bis-methacrylamide, glycidyl methacrylate, allyl glycidyl ether
and methacrylamide. Both Eupergit C and Eupergit C 250 L are
microporous, epoxy-activated acrylic beads with a diameter of
100–250 ␮m. They differ in the content of oxirane groups and
in their porosity. While Eupergit C has an average pore size of
r = 10 nm and an oxirane density of 600 ␮mol/g dry beads, Eupergit
C 250 L has larger pores (r = 100 nm) and a lower oxirane density
Hydroxynitrile lyases (HNLs; EC 4.1.2.x) are a group of enzymes
(
mainly plant enzymes) that in vivo promote the reversible cleav-
age of cyanohydrins into hydrocyanic acid (HCN) and aldehyde
or ketone). The release of HCN by the plant – a process called
(
cyanogenesis – presumably serves as a defense mechanism against
herbivores and microbial attack [1]. Despite the natural function
of HNLs as metabolic enzymes-cleavage of C–C bond – they are
powerful in vitro catalysts for the preparation of optically active
cyanohydrins (hydroxynitriles) – a carboligation process. Opti-
cally active (chiral) cyanohydrins are important building blocks
for preparing the fine chemicals, pharmaceuticals, agrochemicals,
veterinary products, vitamins and food additives. The preparation
of hydroxynitriles with high optical purity necessitates a very fast
enzymatic reaction to ensure that the rate of the spontaneous reac-
tion can be neglected and the enzymatic reaction is the dominant
factor in the formation of hydroxynitrile (Fig. 1).
Recently, HNLs have become a highly appealing tool in the
synthesis of chiral cyanohydrins, because they are cofactor-
independent enzymes, usually show high enantio-, chemo- and
regioselectivities, have wide range of substrates specifities, work
(300 ␮mol/g dry beads) [3]. Because of their structure, Eupergit C
and Eupergit C 250 L are stable, both chemically and mechanically,
over a pH range from 0 to 14, and do not swell or shrink even upon
drastic pH changes in this range. Thus, they have been described as
a suitable carriers for enzymes in enzyme immobilization [2–6]. In
∗ Corresponding author. Tel.: +90 322 3386081/26; fax: +90 322 3386070.
E-mail addresses: stukel@cu.edu.tr (S.S. Tükel), dozyildirim@gmail.com
D. Yildirim), alagozdilek@yahoo.com (D. Alagöz), alptekinozlem@yahoo.com
Ö. Alptekin), gybilgic@cu.edu.tr (G. Yücebilgi c¸ ), rbilgin@cu.edu.tr (R. Bilgin).
(
(
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.05.001

1
62
S.S. Tükel et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 161–165
phosphate buffer (pH 7.0). The reaction was allowed to continue for
◦
2
4 h at 25 C. The mixture was shaken gently during immobilization
period in a shaken water bath. After that, the resulting immobilized
HNL was washed extensively with distilled water until no protein
◦
was detected in the filtrate, then stored at 5 C. The protein con-
tents of the solutions were determined by the method of Lowry et
al. [9]. BSA was used as a standard protein. The amount of immobi-
lized enzyme protein was estimated by subtracting the amount of
protein determined in the filtrate from the total amount of protein
used in immobilization procedure.
2
2
.2.3. Lyase activity of HNL
.2.3.1. Lyase activity assay of HNL. Lyase activity of HNL was
determined spectrophotometrically by measuring the amount of
benzaldehyde from rac-MN. Briefly, 2.85 mL of acetate buffer
(
50 mM, pH 6.0) and 0.1 mL of 1 mg/mL free enzyme or 10 mg
Fig. 1. General synthesis of cyanohydrins: 1, enzymatic reaction; 2, spontaneous
reaction.
immobilized HNL were incubated at room temperature for 2 min.
The reaction was started by the addition of 0.05 mL of 30 mM
rac-MN solution as substrate prepared in 95% of ethanol. At the
end of 10 min, 0.5 mL of aliquots taken from the reaction mixture
was diluted to 3 mL with 50 mM acetate buffer (pH 4.0) and its
absorbance at 250 nm was measured immediately against blank
solution containing 3 mL of 50 mM acetate buffer (ODe).
this study, we attempted the isolation, partial purification, charac-
terization of HNL from seeds of Prunus pseudoarmeniaca and then
immobilization of HNL onto Eupergit C and Eupergit C 250 L. Also,
we investigated the synthesis of (R)-mandelonitrile by using free
and immobilized HNL preparations.
Two controls were run in parallel one 2.95 mL acetate
buffer + 0.05 mL substrate solution (OD ) and another 2.90 mL
b
2
. Materials and methods
acetate buffer + 0.10 mL enzyme solution (ODp) and their
absorbances were measured at 250 nm described above.
HNL activity was calculated from the equation,
2.1. Materials
ꢀ
OD
Vt
Ripened P. pseudoarmeniaca fruits (a wild apricot) were obtained
Activity (U/ml) =
×
ꢀOD = ODe − (OD + ODp)
b
ε × t
Ve
from C¸ ukurova University, Agricultural Department. Eupergit C
and Eupergit C 250 L, racemic mandelonitrile (rac-MN), ben-
zaldehyde, sodium cyanide (NaCN), dimethylsulfoxide (DMSO),
tert-buthylmethylether (TBME), acetone, pyridine, acetic anhy-
dride, bovine serum albumin (BSA) and all other reagents and
solvents were purchased from Sigma (St. Louis, MO).
where Vt and Ve are total volume of the reaction solution and
enzyme volume used in the reaction, respectively. ε is absorption
coefficiency of benzaldehyde (18,777 M cm ) at 250 nm, t is the
−1
−1
reaction time (min).
2
.2.3.2. Effect of pH onto lyase activity of HNL. The effect of pH on
2
2
.2. Methods
the activities of free and immobilized HNL preparations was deter-
mined in 50 mM acetate buffer (pH 5.0 and 5.5), citrate buffer (pH
6
.2.1. Partial purification of HNL
Ripened wild apricot fruits were taken and the fleshy covers
.0) and phosphate buffer (pH 6.5 and 7.0).
of its were removed. The upper layer of seeds was cracked with
a hammer to obtain soft kernel inside. About 200 g of kernels
2
.2.3.3. Effect of buffer concentration onto lyase activity of HNL. The
effect of buffer concentration on the activities of free and immobi-
lized HNL preparations was studied in 25, 50, 75 and 100 mM buffer
solutions at predetermined optimum pH.
◦
were homogenized in 200 mL of cold acetone (−20 C) by using a
prechilled Waring blender for 2 min at maximum speed. The slurry
was filtered and the residue was extracted with 200 mL of cold ace-
tone. After discarding the solvent each time this procedure was
repeated until a white powder was obtained. The resulting powder
2
.2.3.4. Effect of temperature onto lyase activity of HNL. The effect of
◦
temperature on enzyme activity was investigated in the range of
1
free and immobilized HNLs.
was dried overnight at room temperature and stored at −20 C until
◦
5–50 C at their optimum pH and buffer concentrations for both
use [7]. In order to obtain enzyme extract, 5 g of the powder was
suspended in 50 mL of prechilled 50 mM phosphate buffer (pH 6.2)
◦
and then stirred for 1 h at 4 C. The suspension was centrifuged at
◦
2.2.3.5. Kinetic parameters for lyase activity of HNL. The enzyme
activity was measured at different substrate concentrations rang-
ing from 0.5 to 5.0 mM at predetermined optimum conditions. Vmax
and Km values of free and immobilized HNLs were calculated from
double reciprocal plot of Lineweaver–Burk.
1
0,000 rpm for 15 min at 4 C. After that, the supernatant as crude
enzyme extract was fractionated with (NH ) SO . The precipitate
4
2
4
fractionated at 40% saturation was collected by centrifugation at
◦
1
3,000 rpm for 20 min at 4 C. The resulting precipitate was dis-
solved in minimum volume of 50 mM phosphate buffer (pH 6.2),
dialyzed overnight against an excess volume of the same buffer
◦
and the solution was stored at 4 C and used as HNL solution. The
2.2.3.6. Thermal stability for lyase activity of HNL. To investigate
protein contents of the solutions were determined by the method
of Bradford [8]. BSA was used as a standard protein.
the thermal stabilities of free and immobilized HNLs, HNL samples
were incubated at 25 and 50 C for different incubation times (1, 2,
◦
4
, 8 and 15 h) and then their residual activities were measured as
2.2.2. Immobilization of HNL
described in Section 2.2.3.1.
The immobilization procedure has been described earlier by
Mateo et al. [6]. Briefly, 1 g of Eupergit C or Eupergit C 250 L was
mixed with 9 mL of HNL solution (1 mg prot./mL) prepared in 1 M
2.2.3.7. Operational stability of immobilized HNL for the lyase activity.
Operational stability of each immobilized HNL was investigated by

S.S. Tükel et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 161–165
163
Fig. 2. The effect of pH on the lyase activities of free and immobilized HNLs.
Fig. 4. The effect of temperature on the lyase activities of free and immobilized
HNLs.
using a batch type column reactor (1.1 × 5 cm). Twenty-five mil-
ligrams of immobilized HNL sample was loaded into the column
reactor and 3 mL substrate solution was added. The reaction solu-
tion was allowed to continue for 10 min. The reaction solution was
then immediately separated from the immobilized HNL sample
and the amount of benzaldehyde produced was determined by the
method as described before. The same experiment using the same
immobilized HNL sample was repeated 20 times in every 10 min.
was determined in 400 mM citrate buffer at different pH values
◦
(
4.0–6.0) at 5 C.
2.2.4.3. Effect of temperature onto carboligation activity of HNL. To
investigate the optimum temperature, carboligation activities of
◦
free and immobilized HNLs were measured in the range of 5–20 C
in 400 mM citrate buffer (pH 4.0).
2
2
.2.4. Carboligation activity of HNL
.2.4.1. Enantioselective synthesis of mandelonitrile by HNL. Two
2.2.4.4. Operational stability of immobilized HNL for the carboliga-
hundred microliters of free HNL or 50 mg of immobilized HNLs
were mixed with 600 ␮L of 400 mM citrate buffer (pH 4.0), 100 ␮L
of 1.0 M benzaldehyde in DMSO and 1.0 mL of TBME. The reac-
tion was started by the addition of 200 ␮L 1.0 M HCN solution in
TBME prepared according to Bhunya et al. [10] and performed on
tion activity. Operational stabilities of immobilized HNLs in terms
of lyase activity were determined by using the same immobilized
HNL sample by 10 successive 24 h reactions in standard assay
conditions.
◦
a shaker for 24 h at 5 C. During the reaction, the reaction mixture
was agitated at 100 rpm. After completion of the reaction, 100 ␮L
3. Results and discussions
of samples were withdrawn and diluted to 0.5 mL with acylation
In this study, HNL was partially purified from seeds of P. pseu-
solution containing 25 ␮L pyridine and 25 ␮L acetic anhydride in
◦
doarmeniaca. HNL was obtained as 4.4-fold purity extract after
0% (NH ) SO precipitation and immobilized onto Eupergit sup-
1
0 mL CH Cl and then was heated at 50 C for 30 min. A hun-
2
2
4
4
2
4
dred microliters of samples were withdrawn and diluted to 0.5 mL
with mobile phase. The yield and ee of samples were analyzed
by HPLC equipped with a UV detector and ORPak CDC 453-HQ
chiral column (4.6 × 150 mm) at 220 nm. Mobile phase was ammo-
nium acetate buffer:acetonitrile (60:40, v/v; pH 4.0), flow rate was
ports. The amounts of bound protein onto per gram of Eupergit
C and Eupergit C 250 L were determined as 7.3 and 8.8 mg
of the initial total amount of protein (9 mg), respectively after
immobilization.
The effect of pH on the lyase activity of free and immobi-
lized HNLs was studied in the pH range of 5.0–7.0 because of the
base-catalyzed decomposition of MN, pH dependence was only
determined below pH 7.5. As shown in Fig. 2, free and immobi-
lized HNLs both showed their maximum activities at the same pH
value as 6.0. Lyase activities of free and immobilized HNLs were
measured at buffer concentration range of 25–100 mM at pH 6.0
and the results were shown in Fig. 3. The optimum buffer concen-
tration of all enzyme preparations were determined as 50 mM. In
the previous studies, the optimum pH values for free HNLs isolated
from other Prunus species were reported in the range of 5.0–7.0
0
.250 mL/min. The spontaneous reaction rate was also determined
by carrying out the reaction under identical conditions without
enzyme.
2.2.4.2. Effect of pH onto carboligation activity of HNL. The effect of
pH on the carboligation activities of free and immobilized HNLs
[
11–14].
Lyase activities of free and immobilized HNLs were measured
◦
at 15–50 C. The optimum temperature of all enzyme prepara-
tions were determined as 25 C (Fig. 4). Free and immobilized HNLs
showed similar behaviour at 15–40 C. However, free HNL retained
about 57% of its maximum activity at 50 C whereas immobilized
◦
◦
◦
HNLs onto Eupergit C and Eupergit C 250 L were retained about 75
◦
and 82% of their maximum activities, respectively at 50 C. In the lit-
erature, the optimum temperature for Eriobotrya japonica (loquat)
HNL [15] and Phlebodium aureum HNL [14] were both reported as
◦
4
0 C by using acetone cyanohydrin as substrate.
The kinetic parameters were determined in 50 mM citrate
buffer, pH 6.0 at 25 C for all enzyme preparations. The maximum
Fig. 3. The effect of buffer concentration on the lyase activities of free and immobi-
lized HNLs.
◦

1
64
S.S. Tükel et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 161–165
Table 1
Effect of pH onto carboligation activity.
pH 4.0
pH 5.0
pH 6.0
Yield (%)
ee (%)
Yield (%)
ee (%)
Yield (%)
ee (%)
Free HNL
100
100
100
99
99
99
63
74
72
90
90
90
30
32
25
80
80
80
Immobilized HNL onto Eupergit C
Immobilized HNL onto Eupergit C 250 L
Table 2
Effect of temperature onto carboligation activity.
◦
◦
◦
5
C
10
C
20
C
Yield (%)
ee (%)
Yield (%)
ee (%)
Yield (%)
ee (%)
Free HNL
100
100
100
99
99
99
96
89
85
99
99
99
74
87
83
99
99
99
Immobilized HNL onto Eupergit C
Immobilized HNL onto Eupergit C 250 L
◦
activity of free HNL was determined as 0.54 U/mg prot. and maxi-
mum activities of immobilized HNLs onto Eupergit C and Eupergit C
bilized enzyme. The half lives (t1/2) of free HNL at 25 and 50 C were
49.9 and 30.5 h, respectively and these correspondingly were 96.3
and 43.6 h for immobilized HNL onto Eupergit C; 138.6 and 50.2 h
for immobilized HNL onto Eupergit C 250 L. The thermal inactiva-
2
50 L were determined as 0.87 and 0.35 U/mg prot., respectively. Km
values were found as 2.23, 1.60 and 1.03 mM for free HNL, immobi-
lized HNL onto Eupergit C and immobilized HNL onto Eupergit C 250
L, respectively. In the previous studies, the Km values of different
purity HNLs were reported as 0.172 mM (for Prunus serotina seeds),
◦
−2
tion rate constants (k ) at 25 and 50 C were calculated as 1.4 × 10
i
−
−1
2
−1
−3
and 2.3 × 10
h
, respectively for free HNL; as 7.2 × 10
and
−
2
1.6 × 10
and; as 5.0 × 10 and 1.4 × 10
h
, respectively for immobilized HNL onto Eupergit C
−
3
−2 −1
h , respectively for immobilized
0
.290 mM (for Prunus dilcus seeds), 0.790 mM (for Sorghum bicolor
seeds) and 0.093 mM (for Prunus lyonii seeds) for mandelonitrile
12,13].
The thermal stability of enzymes is very important parameter
HNL onto Eupergit C 250 L. These results showed that the thermal
stabilities of immobilized HNLs were comparably higher than that
of the free HNL at both temperatures. The high thermal stabilities
of immobilized HNLs onto Eupergit C and Eupergit C 250 L as com-
pared to that of free HNL may suggest the formation of multipoint
covalent attachments between a high proportion of the enzyme
molecule and Eupergit supports.
[
in industrial process. It is often observed that immobilized enzyme
has higher thermal stability than the corresponding free enzyme
because of the reduction of conformational flexibility in the immo-
The reuse numbers of immobilized enzymes are one of the most
important aspects for industrial application. An increased stabil-
ity could make the immobilized enzymes more advantageous than
their free form. Operational stabilities of immobilized HNLs for
lyase activity were determined in batch type column reactor. As
shown in Fig. 5, both of immobilized HNLs were used repeatedly
2
0 times and the residual activities were about 97% of their initial
activities.
The carboligation activities of free and immobilized HNLs were
also evaluated for (R)-mandelonitrile (R-MN) synthesis from ben-
zaldehyde and HCN. The optical configuration of synthesized R-MN
was identified by comparing the retention time of synthesized R-
MN with that of the optically active standard compounds. The
retention times of S-MN and R-MN were 4.82 and 6.22 min, respec-
tively.
The pH profiles of the carboligation activities of free and immo-
bilized HNLs were investigated at different pHs and the results were
Fig. 5. The operational stabilities of immobilized HNLs for lyase activity.
Fig. 6. The operational stabilities of immobilized HNLs for carboligation activity (×): yield %, (ꢀ): ee%. (a) Immobilized HNL onto Eupergit C. (b) Immobilized HNL onto
Eupergit C 250 L.

S.S. Tükel et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 161–165
165
given in Table 1. The maximum yield and ee% were determined at
pH 4.0 for all HNL preparations. It was found that increasing the pH
value above 4.0, the yield and ee were dramatically decreased due
to the spontaneous chemical decomposition of the product.
As shown from Table 2, all HNL preparations showed optimal
(b) Eupergit C and Eupergit C 250 L are suitable carriers for
HNL immobilization. The thermal stability and reusability of
HNL were improved upon immobilization onto Eupergit C and
Eupergit C 250 L.
◦
carboligation activity at 5 C. As the temperature was increased
Acknowledgement
◦
from 5 to 20 C the yields decreased however ee values (99%)
unchanged. In the literature, Nanda et al. [16] investigated the
enantioselective synthesis of cyanohydrins using partially puri-
fied free HNL from Prunus mume and the yield and ee of R-MN
were determined as 65 and 95%, respectively. Ueatrongchit et
This work was supported by TUBITAK (The Scientific and Tech-
nical Research Council of Turkey) with the Project number of
109T427.
◦
al. [17] reported that the optimum temperature was 10 C for
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. Conclusions
The present study demonstrates that
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(
a) The P. pseudoarmeniaca is a good source of HNL and P. pseu-
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(
R)-mandelonitrile.