Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat)

Article abstract of DOI:10.1271/bbb.80023

A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH₄)₂SO₄ fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K_m of 161 μM and a k _cat/K_m of 348 s^-1 mM^-1 for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40°C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO₄, HgCl₂, AgNO₃, FeCl₃, β-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k_cat/K_m) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.

Full text of DOI:10.1271/bbb.80023

Biosci. Biotechnol. Biochem., 72 (6), 1513–1522, 2008
Purification and Characterization of A Novel (R)-Hydroxynitrile Lyase
from Eriobotrya japonica (Loquat)
Techawaree UEATRONGCHIT,1 Ai KAYO,2 Hidenobu KOMEDA,2
y
y
1;
Yasuhisa ASANO,2; and Aran H-KITTIKUN
1Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University,
Hat Yai, Songkhla 90112, Thailand
2Biotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan
Received January 10, 2008; Accepted March 13, 2008; Online Publication, June 7, 2008
[doi:10.1271/bbb.80023]
A hydroxynitrile lyase was isolated and purified to
homogeneity from seeds of Eriobotrya japonica (loquat).
The final yield, of 36% with 49-fold purification, was
obtained by 30–80% (NH4)2SO4 fractionation and
column chromatography on DEAE-Toyopearl and Con-
canavalin A Sepharose 4B, which suggested the presence
of a carbohydrate side chain. The purified enzyme was a
monomer with a molecular mass of 72 kDa as deter-
mined by gel filtration, and 62.3 kDa as determined by
SDS-gel electrophoresis. The N-terminal sequence is
reported. The enzyme was a flavoprotein containing
FAD as a prosthetic group, and it exhibited a Km of
of scientists and industry. Chiral cyanohydrins, alcohols
containing a cyano group attached to the same carbon
atom, are versatile building blocks in the synthesis of
large numbers of biologically active compounds in fine
chemicals, pharmaceuticals, veterinary products, crop-
protecting agents, vitamins, and food additives.2) HNL
activity was detected for the first time in kernels of
Prunus dulcis (amygdalus) or almond by Friedrich
Wohler in 1837, cleaving the cyanohydrins to aldehyde
¨
and HCN.3) The synthesis of chiral cyanohydrins by
HNL from almond was first reported by Rosenthaler.4)
Recently, we developed a new screening method
using chiral HPLC to determine the activity and stereo-
selectivity of HNL.5) We discovered several novel
sources of HNL among the 163 plant species in 74
families examined. (S)-HNL activity was found in a
homogenate of the leaves of Baliospermum montanum,
while (R)-HNLs were detected in homogenates of the
leaves and seeds of Passiflora edulis, and the seeds of
Eriobotrya japonica, Chaenomles sinensis, Sorbus au-
cuparia, Prunus mume, and Prunus persica.
À1
161 ꢀM and a kcat=Km of 348 sÀ1 mM for mandeloni-
trile. The optimum pH and temperature were pH 5.5
and 40 ꢀC respectively. The enzyme showed excellent
stability with regard to pH and temperature. Metal ions
were not required for its activity, while activity was
significantly inhibited by CuSO4, HgCl2, AgNO3, FeCl3,
ꢀ-mercaptoethanol, iodoacetic acid, phenylmethylsulfo-
nylfluoride, and diethylpyrocarbonate. The specificity
constant (kcat=Km) of the enzyme was investigated for
the first time using various aldehydes as substrates.
The enzyme was active toward aromatic and aliphatic
aldehydes, and showed a preference for smaller sub-
strates over bulky one.
Eriobotrya japonica (Thunb.) Lindley, also known as
Japanese medlar, is an evergreen tree of the Maloideae
subfamily of the Rosaceae family that is native to
southeastern China, long since introduced to Japan,
hence its name. It is cultivated as well in tropical and
subtropical countries.6) Its golden fruit, the loquat, is
round or oval in shape and has a sweet taste. The fruit is
eaten fresh or is made into preserves, jam, jelly, juice,
and nectar after removal of the seeds as waste.7) The
seeds of Eriobotrya L. have been reported to be a source
of (R)-hydroxynitrile lyase for the synthesis of cyano-
hydrins,8) but research on the enzymatic synthesis of
chiral cyanohydrins synthesis has been carried out
mostly using an excess amount of crude enzyme and a
long reaction time to obtain high enantiomeric excess
(e.e.) and conversion. Crude enzyme or seed powder has
Key words: hydroxynitrile lyase; mandelonitrile lyase;
Eriobotrya japonica; flavoprotein; cyanohy-
drins
Hydroxynitrile lyase (HNL) is one of the enzymes
involved in the catabolism of cyanogenic glycosides in
higher plants. This enzyme catalyzes the decomposition
of cyanohydrins into the corresponding aldehydes or
ketones and HCN for plant defense against predators and
microorganisms.1) The reverse reaction of HNL, syn-
thesis of chiral cyanohydrins, has attracted the attention
y
To whom correspondence should be addressed. Yasuhisa ASANO, Fax: +81-766-56-2498; Tel: +81-766-56-7500 ext 530; E-mail: asano@
pu-toyama.ac.jp; Aran H-KITTIKUN, Fax: +66-7421-2889; Tel: +66-7428-6363; E-mail: aran.h@psu.ac.th
Abbreviations: HNL, hydroxynitrile lyase; EjHNL, hydroxynitrile lyase from Eriobotrya japonica

1514
T. UEATRONGCHIT et al.
been used directly for the reaction without purification
and characterization of the enzyme.8–10) In the case of
HNL from E. japonica (EjHNL), lack of information
and understanding of the characteristics of purified
enzyme have led to limited enzyme applications.
To understand the characteristics of EjHNL, we
attempted the isolation, purification, and characteriza-
tion of HNL from seeds of E. japonica and its
application in the synthesis of cyanohydrins.
with 10 mM potassium phosphate buffer, pH 6.0, and
eluted with a linear gradient of NaCl (0–0.5 M).
Fractions of 2 ml were collected. The protein profile
was monitored by measuring absorbance at 280 nm,
the protein fractions were assayed for HNL activity, and
the active fractions were pooled for further analysis.
Concanavalin A Sepharose 4B column chromatogra-
phy. The enzyme solution was purified on a Concana-
valin A Sepharose 4B column (5 mm ꢁ 5 cm, 1 ml)
equilibrated with 0.5 M NaCl in 10 mM potassium
phosphate buffer, pH 6.0, and eluted with linear gradient
of 0–1 M of ꢂ-D-methylglucoside. Fractions of 1 ml were
collected. The protein profile was monitored by meas-
uring the absorbance at 280 nm, and the protein fractions
were assayed for HNL activity.
Materials and Methods
Materials. Seeds of E. japonica were purchased from
the National Federation of Agricultural Cooperative
Associations (Nagasaki, Japan) and stored at 4 ꢀC. All
chemicals used in the experiments were purchased from
commercial sources and were used without further
purification. Silica gel TLC plates (Merck, NJ) were
used to follow the chemical reaction. Silica gel 100–200
mesh (Wako Pure Chemical Industries, Osaka) was used
in column chromatography. NMR spectra were recorded
with a JEOL LA-400 spectrometer (Tokyo) at 25 ꢀC in
CDCl3 using TMS as the internal standard. Chemical
Activity assay. HNL activity was measured by
monitoring the decomposition of (R,S)-mandelonitrile
to benzaldehyde by the method described by
Willeman,11) with a slight modification. The conversion
of (R,S)-mandelonitrile to benzaldehyde was followed
by continuously measuring the increase in absorbance
at 280 nm. The reaction was performed in a quartz
cell. The enzyme solution was added to a 50 mM
sodium citrate phosphate buffer, pH 5.5, containing
2 mM mandelonitrile in a total volume of 1 ml. Then the
reaction mixture was mixed gently, and the reaction was
followed for 5 min by spectrophotometer at 280 nm
(U-3210 spectrophotometer, Hitachi, Tokyo). The linear
change in absorbance for the initial 1 min was used in
the calculation. The slope of absorbance was determined
1
shifts were shown as ꢀH and ꢀC for H and 13C NMR
spectra respectively. Chiral HPLC was performed using
a chiralcel OJ-H column (Diacel Chemical Industries,
Osaka) with a SPD-10A VP UV-vis detector (Shimadzu,
Kyoto) at 254 nm. The eluting solvent was 90% hexane
and 10% isopropanol by volume. A chiral GC analysis
was performed using a Gas Chromatograph GC-14B
(Shimadzu) with a fused silica capillary ꢁ-Dex-325
column (Supelco, PA) and He as a carrier gas (Detector
temperature, 230 ꢀC; injection temperature, 220 ꢀC).
and subtracted with a blank. By "280 ¼ 1:4 mMꢂ1 cmꢂ1
,
the enzyme activity was calculated.
Crude enzyme extraction. Seeds of Eriobotrya japon-
ica were sterilized by soaking in 0.1% (v/v) sodium
hypochlorite and rinsed with de-ionized water. All
purification steps were carried out at 4 ꢀC. Sterilized
seeds were homogenized with 3% polyvinylpyrolidone
in 10 mM potassium phosphate buffer, pH 6.0 (100 ml/
100 g of fresh seeds) in a SMT Process Homogenizer,
PH91 (SMT, Tokyo). The homogenate was filtered
through four layers of cheesecloth and centrifuged at
28;000 ꢁ g for 30 min. The supernatant was used as
the crude enzyme extract.
One unit of HNL activity (1 decomposition unit)
was defined as the amount of enzyme that converted
1 mmol of mandelonitrile to benzaldehyde in 1 min under
standard assay conditions. In a previous study of EjHNL
by our group, 1 unit of HNL activity (1 synthetic unit)
was defined as the amount of enzyme that produced
1 mmol of optically active mandelonitrile from benzal-
dehyde per min under the described assay conditions.5)
The decomposition activity of EjHNL was about 1.3
times more than the synthetic activity.
Protein assay. Protein concentrations were measured
using a Bio-Rad protein assay kit with BSA as a
standard (Bio-Rad Laboratories, Hercules, CA).12)
Ammonium sulfate precipitation. The crude extract
was fractionated by 0–30, 30–80% saturation of ammo-
nium sulfate. The precipitate fractionated at 30–80%
was collected by centrifugation at 28;000 ꢁ g for
30 min. The precipitate was dissolved and dialyzed
overnight against an excess volume of the same buffer.
Precipitate formed during dialysis was removed by
centrifugation, and the supernatant was collected.
Gel electrophoresis. SDS–PAGE and Native PAGE
were used to analyze the molecular mass and homoge-
neity of the enzyme respectively. Gel electrophoresis
was performed with a 1-mm thick polyacrylamide gel.13)
Standard protein markers for SDS–PAGE consisted of
phosphorylase b (97,400 Da), bovine serum albumin
(66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase
(31,000 Da), soybean trypsin inhibitor (21,500 Da), and
lysozyme (14,400 Da).
DEAE-Toyopearl 650M column chromatography. The
enzyme solution was loaded onto a DEAE-Toyopearl
650M column (26 mm ꢁ 10.5 cm, 50 ml) equilibrated

Purification and Characterization of EjHNL
1515
Estimation of apparent molecular mass. The molecu-
lar mass of EjHNL was estimated by gel-filtration
chromatography on a Superdex-200 column HR 10/30
(10 mm ꢁ 30 cm, 24 ml). The purified enzyme was
loaded onto the column and eluted at a flow rate of
0.2 ml/min with 10 mM potassium phosphate buffer,
pH 6.0, containing 0.15 M NaCl. The standard protein
markers used for calibration were as follows: thyroglo-
bulin (670,000 Da), ꢃ-globulin (158,000 Da), ovalbumin
(44,000 Da), myoglobin (17,000 Da), and vitamin B2
(13,500 Da).
the conditions described above at various aldehyde
substrate concentrations.
Preparation of cyanohydrins for HPLC and GC
standard. The carbonyl compound (1 eq.) was dissolved
and stirred vigorously in acetic acid. This was followed
by the addition of an aqueous solution of KCN (3 eq.)
into the mixture. The reaction was monitored by TLC
with UV light or iodine vapor as a developing agent.
After the reaction was completed, the reaction mixture
was neutralized with NaHCO3, extracted with ethyl
acetate, dried with anhydrous Na2SO4, and evaporated
in a vacuum. The cyanohydrins after purification by
silica gel column chromatography were characterized
by 1H and 13C-NMR spectroscopy and used as standards
for HPLC and GC. All cyanohydrins isolated were
colorless to slightly yellow oils.
N-Terminal sequence. The N-terminal sequence of the
purified enzyme (40 ml) was sequenced (30 cycles) with
a HP G1005A Protein Sequencing system from APRO
Science (Tokushima, Japan).
Prosthetic group analysis. Absorption spectra were
measured with a PharmaSpec UV-1700 spectrophotom-
eter (Shimadzu, Kyoto, Japan), using a 1-cm cell path
length. The enzyme was analyzed in 10 mM potassium
phosphate buffer, pH 6.0.
NMR data for standard cyanohydrins.
2b: 2-hydroxy-2-(4-methoxyphenyl)acetonitrile (p-
anisaldehyde cyanohydrin)
1
C9H9O2N; H NMR ꢀH (CDCl3): 7.4 (d, 2H, Ar-H),
6.9 (d, 2H, Ar-H), 5.4 (d, 1H, CHCNOH), 3.8 (s, 3H,
CH3O-Ar), 2.6 (d, 1H, OH). 13C NMR ꢀC (CDCl3):
160.8, 128.3, 127.5, 118.8, 114.5, 63.4, 55.4
2c: 2-hydroxy-2-(thiophen-2-yl)acetonitrile (2-thio-
phene carboxaldehyde cyanohydrin)
C6H5ONS; 1H NMR ꢀH (CDCl3): 7.0–7.5 (m, 3H, Ar-
H), 5.7 (s, 1H, CHCNOH), 2.7 (s, 1H, OH). 13C NMR
ꢀC (CDCl3): 148.0, 128.1, 127.4, 127.2, 118.2, 59.36
2d: 2-hydroxy-2-(naphthalen-1-yl)acetonitrile (1-
naphthaldehyde cyanohydrin)
C12H9ON; 1H NMR ꢀH (CDCl3): 7.5–8.1 (m, 7H, Ar-
H), 6.1 (d, 1H, CHCNOH), 2.7 (d, 1H, OH). 13C NMR
ꢀC (CDCl3): 134.1, 131.1, 130.4, 130.0, 129.1, 127.4,
126.6, 125.7, 125.1, 122.9, 118.0
Kinetic parameters. The initial velocity of the
enzymatic decomposition of racemic mandelonitrile
was determined in 50 mM sodium citrate buffer,
pH 5.5, according to the standard reaction assay at
various substrate concentrations.
Effects of pH and temperature. The optimum pH
and temperature for activity were assayed according to
the standard method at pH 3.5–6.5 and temperatures of
10–80 ꢀC. Stability was monitored after 60 min incuba-
tion at various pH levels (3–9, 30 ꢀC) and temperatures
(0–80 ꢀC, pH 6.0).
Effect of additives. The effects of various additives on
the purified enzyme were examined. The enzyme
solution was incubated with various additives in 10
mM potassium phosphate buffer, pH 6.0, at 30 ꢀC for
60 min. The remaining activity of the enzyme was
assayed according to standard procedures.
2e: 2-hydroxy-2-(naphthalen-2-yl)acetonitrile (2-
naphthaldehyde cyanohydrin)
C12H9ON; 1H NMR ꢀH (CDCl3): 7.4–8.0 (m, 7H, Ar-
H), 6.0 (d, 1H, CHCNOH), 2.6 (d, 1H, OH). 13C NMR
ꢀC (CDCl3): 135.2, 133.7, 131.8, 128.0, 127.6, 127.5,
127.3, 127.0, 126.0, 125.1, 118.2, 63.3
2f: 2-(benzo[d][1,3]dioxol-6-yl)-2-hydroxyacetoni-
trile (piperonaldehyde cyanohydrin)
Synthesis of cyanohydrins. The reaction mixture was
prepared in a micro-tube: 1.0 M of carbonyl compound
(in DMSO, 40 ml) was added to sodium citrate buffer
(400 mM, pH 4.0, 760 ml), followed by the addition of
enzyme solution (25 units, decomposition units) and
KCN solution (1.0 M, 100 ml). The reaction was moni-
tored by taking a small aliquot of the reaction mixture
(100 ml) and extraction with organic solvent (90%
n-hexane, 10% isopropanol by volume for HPLC, ethyl
acetate for GC). The organic layer was analyzed by
chiral HPLC (for aromatic compounds) and chiral GC
(for aliphatic compounds).
C9H7O3N; 1H NMR ꢀH (CDCl3): 7.0 (dd, 2H, Ar-H),
6.8 (dd, 1H, Ar-H), 6.0 (s, 2H, -OCH2O-), 5.4 (d, 1H,
CHCNOH), 2.6 (d, 1H, OH). 13C NMR ꢀC (CDCl3):
148.9, 148.4, 129.1, 120.7, 118.6, 108.6, 107.2, 101.6,
63.5
2g: 2-hydroxybutanenitrile (propionaldehyde cyano-
hydrin)
C4H7ON; 1H NMR ꢀH (CDCl3): 4.4 (t, 1H,
CHCNOH), 2.9 (s, 1H, OH), 1.8 (qd, 2H, CH2), 1.1 (t,
3H, CH3). 13C NMR ꢀC (CDCl3): 119.8, 62.5, 28.6, 8.9
2h: 2-hydroxy-3-methylbutanenitrile (isobutyralde-
hyde cyanohydrin)
To calculate the kinetic parameters of the enzymatic
synthesis of cyanohydrins, the initial velocity of the
synthesis of chiral cyanohydrins was determined under
C5H9ON; 1H NMR ꢀH (CDCl3): 4.3 (m, 1H,
CHCNOH), 2.8 (s, 1H, OH), 2.1 (m, 1H, CH), 0.9 (m,

1516
T. UEATRONGCHIT et al.
Table 1. Purification of Hydroxynitrile Lyase from Eriobotrya japonica
Total activity
(Units)
Total protein
(mg)
Specific activity
(Units/mg)
Yield
(%)
Purification
(fold)
Purification step
Crude extract
30–80% (NH4)2SO4
840
522
369
303
1000
140
38.3
7.5
0.8
3.7
100
62
1
4
DEAE-Toyopearl 650M
Concanavalin A Sepharose 4B
9.7
40.9
44
36
12
49
6H, CH3). 13C NMR ꢀC (CDCl3): 118.4, 67.0, 33.0,
17.6, 17.1
1
2i: 2-hydroxy-3,3-dimethylbutanenitrile (pivalalde-
hyde cyanohydrin)
A
C6H11ON; 1H NMR ꢀH (CDCl3): 4.1 (d, 1H,
CHCNOH), 2.4 (d, 1H, OH), 1.0 (s, 9H, CH3).
13C NMR ꢀC (CDCl3): 118.4, 70.7, 35.4, 24.9
2j: 2-cyclohexyl-2-hydroxyacetonitrile (cyclohexane-
carboxaldehyde cyanohydrin)
C8H13ON; 1H NMR ꢀH (CDCl3): 4.2 (t, 1H,
CHCNOH), 2.5 (d, 1H, OH), 1.8 (m, 10H, CH2), 1.2
(m, 1H, CH). 13C NMR ꢀH (CDCl3): 119.2, 66.4, 42.2,
30.9, 28.1, 27.7, 25.9, 25.4
Results
A hydroxynitrile lyase from seeds of Eriobotrya
japonica (EjHNL) was purified to homogeneity. The
results for a typical preparation are summarized in
Table 1.
1
M
Da
B
Crude enzyme with 840 units of activity was
extracted from 2 kg of seeds. Ammonium sulfate
precipitation and salting out were used to concentrate
the crude enzyme solution before column chromatog-
raphy. EjHNL was precipitated between 30 and 80%
saturated salt solution. Approximately 62% of the
activity remained, and 4-fold purification was obtained
by the salt precipitation step.
97,400
66,200
45,000
31,000
The re-dissolved enzyme solution was further purified
by anion-exchange chromatography on a column of
DEAE-Toyopearl 650M. Proteins absorbed on the
column were eluted by increasing the linear gradient
from 0 to 0.5 M NaCl. Fractions having HNL activity
were combined for further purification. Specific activity
increased approximately 3-fold.
An active pool of DEAE-Toyopearl fractions was
dialyzed against 10 mM potassium phosphate buffer,
pH 6.0. Prior to being loaded onto the affinity column,
Concanavalin A Sepharose 4B, the enzyme solution was
mixed with NaCl to obtain a concentration of 0.5 M.
After loading and washing, the column was eluted
with 0–1 M of ꢂ-D-methylglucoside as a linear gradient.
One major symmetrical peak of protein was eluted
at approximately 0.2 M of ꢂ-D-methylglucoside. Large
amounts of contaminated proteins were removed. By
this step, purification 49-fold with a yield of 36% was
obtained. The enzyme was interacted with a concana-
valin A column, indicating that it was a glycoprotein.
The native molecular mass of purified EjHNL was
21,500
14,400
Fig. 1. Native-PAGE (a) and SDS–PAGE (b) of Purified EjHNL.
M, Low molecular weight standard marker; lane 1, purified
EjHNL.
estimated by HPLC on a Superdex 200 filtration column.
From logarithmic plots of molecular mass vs. retention
time of standard proteins, the molecular mass of the
native enzyme was estimated to be 72 kDa. The purified
enzyme appeared as one band on native-PAGE gel
(Fig. 1A) with a molecular mass of 62.3 kDa from the
SDS–PAGE gel (Fig. 1B). Hence, it was concluded
that the purified enzyme consisted of a monomer of a
single subunit. A thick purified band in SDS–PAGE
might have been caused by the glycosidic properties of
the enzyme.

Purification and Characterization of EjHNL
1517
The effect of pH on enzyme activity is shown in
Fig. 3. The optimum pH for EjHNL using the natural
substrate mandelonitrile was found to be approximately
5.5. At pH 3.5, activity was totally inhibited, but a rapid
increase in activity was found at increasing pH levels up
to the optimum. At higher pH than optimum, an increase
in the spontaneous decomposition of the substrate was
observed, and hence the actual optimum pH was difficult
to determine.5) After incubation at various pH for
60 min, excellent stability was found at pH 3–9 (data
not shown). At pH 5.5, the optimum temperature was
40 ꢀC for mandelonitrile as for substrate (Fig. 4).
Temperatures lower and higher than the optimum caused
a regression in activity. Besides the heat-inactivation
effect, high temperatures were found to be a cause
of spontaneous decomposition of the substrate, which
affected the enzymatic activity. Temperature stability
was studied, and EjHNL was found to be active over a
broad range of temperatures (0–60 ꢀC) after incubation
for 60 min. At 70 and 80 ꢀC, the residual activities of the
enzyme after incubation were 68 and 28%, respectively
(data not shown). At 4 ꢀC in 10 mM potassium phosphate
buffer (pH 6.0), EjHNL was stable for at least 30 d
without any significant loss of activity.
Next the influences of various additives on the
activity of EjHNL were determined (Table 2). The
rate of degradation of mandelonitrile due to EjHNL was
unaffected by 1 mM of ZnCl2, MnCl2, MgCl2, or PbCl2,
and by 10 mM of Na2EDTA, while 1 mM of FeCl3 caused
50% inhibition. These results suggest that metal ions
were not required for the activity. The enzyme was
significantly inhibited by a reducing agent and a cysteine
modifying agent. ꢁ-Mercaptoethanol caused 19% inhib-
ition at 10 mM, iodoacetic acid caused 53% inhibition at
1 mM, while CuSO4, HgCl2, and AgNO3 caused com-
plete inhibition. The serine modifying agent phenyl-
methylsulfonylfluoride (PMSF) inhibited the enzymatic
activity by 59% at 1 mM, while the histidine inhibitor
50
40
30
20
10
0
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
1/V
-8
-6
-4
-2
0
2
4
6
8
10
12
1/[S]
0
1
2
3
4
Mandelonitrile (mM)
Fig. 2. Substrate Saturation Curve for Mandelonitrile with EjHNL.
The assay conditions are described in ‘‘Materials and Methods.’’
The concentration of mandelonitrile was varied as indicated. The
inset is a Lineweaver-Burk plot. A Vmax of 46.5 mmol/min/mg, a
Km value of 161 mM, and kcat 56 sꢂ1 and kcat=Km 348 sꢂ1 mM were
ꢂ1
obtained.
The yellow color of EjHNL was also observed during
purification, and the enzyme exhibited maximum ab-
sorption spectra at 278, 389, and 454 nm, as seen in the
absorption spectra of a typical FAD containing protein.
The N-terminal sequence of the purified EjHNL was
L-A-T-P-S-E-H-D-F-S-Y-S-K-S-V-V-X-A-T-D-L-P-Q-
E-E-V-Y-D. X represents an unidentified amino acid.
Fifty-seven percent identity in 28 amino acids was
found to overlap with the sequence of the FAD-
containing (R)-hydroxynitrile lyases from Prunus sero-
tina14) and Prunus dulcis,15) but was different from
non-FAD hydroxynitrile lyases. Therefore, EjHNL was
identified as the FAD-containing HNL.
A substrate saturation curve for purified EjHNL was
determined using mandelonitrile. As shown in Fig. 2,
the Michaelis-Menten kinetics were typical and exhib-
ited a Km of ꢂ1161 mM, a kcat of 56 sꢂ1, and a kcat=Km
of 348 sꢂ1 mM
.
2.5
2
100
80
60
40
20
0
1.5
1
0.5
0
3.0
4.0
5.0
6.0
7.0
pH
Fig. 3. pH Profiles of Purified EjHNL and Spontaneous Decomposition of Mandelonitrile.
, Enzyme activity; , spontaneous decomposition of mandelonitrile.

1518
T. UEATRONGCHIT et al.
100
80
60
40
20
0
3.5
3
2.5
2
1.5
1
0.5
0
0
20
40
60
80
Temperature (oC)
Fig. 4. Temperature Profiles of Purified EjHNL and Spontaneous Decomposition of Mandelonitrile.
, Enzyme activity; , spontaneous decomposition of mandelonitrile.
ꢂ1
s
ꢂ1 mM
respectively. Cyclohexanecarboxaldehyde
Table 2. Effects of Various Additives on the Activity of Purified
EjHNL
was a poor substrate for EjHNL. The enantiomeric
excess (e.e.) of chiral cyanohydrins on different sub-
strates was studied (Table 3). The e.e. was in the range
of 3.9–89.5%. Two factors might have affected the e.e.
value: the specificity of the enzyme for the substrates
used, and the rate of spontaneous chemical reaction.
A high e.e. for chiral cyanohydrins can be achieved
with biphasic systems of buffers and water-immiscible
organic solvents, and the spontaneous chemical reaction
might have been suppressed by these systems.5) These
results will be described elsewhere.
Concentration
(mM)
Inhibition
(%)
Additive
ꢁ-Mercaptoethanol
Iodoacetic acid
PMSF
10
1
19
53
59
36
0
1
DEP
1
Na2EDTA
MnCl2
ZnCl2
10
1
0
1
0
MgCl2
CuSO4
1
0
1
100
100
0
HgCl2
PbCl2
1
1
Discussion
AgNO3
FeCl3
1
100
50
1
HNL was originally classified based on enantioselec-
tivity into two groups. (R)-selective HNL catalyzes
the formation of (R)-cyanohydrins and derived almost
entirely from oxidoreductase ancestors such as HNLs
from Rosaceae and Linum usitatissimum, except that
an (R)-selective HNL from Arabidopsis thaliana is
derived from an ꢂ=ꢁ-hydrolase fold.16,17) (S)-selective
HNL, derived from hydrolases with an ꢂ=ꢁ-hydrolase
fold, catalyzes the formation of (S)-cyanohydrins
such as HNLs from Hevea brasiliensis and Manihot
esculenta.16)
However, according to the EC number based on the
chemical reaction of the enzyme, HNL can be classified
into four groups. First, mandelonitrile lyase (EC
4.1.2.10) is a (R)-HNL that converts the natural substrate
(R)-mandelonitrile to benzaldehyde and prussic acid.
Mandelonitrile lyases from the Rosaceae family (Pru-
noideae and Maloideae subfamilies) are glycoproteins
with posttranslational modifications, and they contain
FAD as a prosthetic group. Several plants containing
this type of HNL have been reported, including Prunus
amygdalus,18) P. serotina,19) P. mume,5) P. persica,5,20)
P. avium,20) P. domestica,20) P. laurocerasus,21) P. lyo-
nii,22) Passiflora edulis,5) Chaenomles sinensis,5) Pou-
teria sapota,20) Cucumis melo,20) Cydonia oblonga,20)
diethylpyrocarbonate (DEP) caused 36% inhibition at
1 mM. These results indicate that cysteine, serine, and
histidine residues are located near the active site of the
enzyme and that they participate in the catalysis.
Synthesis of chiral cyanohydrins from selected ar-
omatic and aliphatic aldehydes (Scheme 1) by EjHNL
was investigated, as shown in Table 3.
A specificity constant (kcat=Km) was used to compare
the relative rate of the enzyme acting on substrates.
For aromatic aldehydes, high specificity constants were
obtained for 2-thiophenaldehyde, benzaldehyde, and ꢂ21-
naphthaldehyde at 160.5, 148.4, and 101.3 sꢂ1 mM
respectively. The specificity constants for bulky aro-
matic substrates, p-aꢂn1isaldehyde and piperonal, were
50.8 and 29.2 sꢂ1 mM respectively, while 1-naphthal-
dehyde was not catalyzed by the enzyme. In contrast to
the report on the substrate specificity of HNL from
Eriobotrya L. by Lin et al.,8) that aliphatic aldehyde
(trimethylacetaldehyde) was an unacceptable substrate
for the enzyme, we found that EjHNL acted on
propionaldehyde, pivaldehyde, and isobutyraldehyde
with specificity constants of 19.4, 14.3, and 9.6

Purification and Characterization of EjHNL
1519
O
C
OH
EjHNL, HCN
C H
R
CN
Buffer, pH 4.0
R
H
1a-j
2a-j
O
C
O
O
C
C
S
H
H
H
H3CO
1a
1b
1e
1c*
O
H
O
O
C
C
C
O
O
H
H
1f
1d
CH3
CH3
H3C
H3C
O
O
O
O
H3C
C
H
H3C
C
H
C
H
C
H
1h
1g
1i
1j
Scheme 1. Synthesis Reaction of Asymmetric (R)-Cyanohydrins Catalyzed by (R)-EjHNL and Aldehyde Substrates.
ꢃ(S)-configuration was assigned according to Cahn-Ingold-Prelog.43)
drin and 2-butanone cyanohydrin, was found in Hevea
brasiliensis27) and Manihot exculenta.28)
Table 3. Cyanohydrin Synthesis and Substrate Specificity of EjHNL
on Aromatic and Aliphatic Aldehydes
Previous studies of the purification and character-
ization of hydroxynitrile lyases investigated only certain
characteristics, such as molecular mass, optimum pH
and temperature, the effects of additives and inhibitors,
storage effects, etc. A description of all characteristics,
including substrate specificity based on activity, was left
incomplete. Seeds of Eriobotrya japonica were found to
be a source of HNL.5) Ground seeds of Eriobotrya L.
were used directly to synthesize chiral cyanohydrins
without further purification and to study the character-
istics of the enzyme.8) Lack of understanding of the true
characteristics of purified enzymes limits their applica-
tion. Therefore, purification and full characterization of
HNL from Eriobotrya japonica (EjHNL) was carried
out, not only for basic studies of the enzyme, but also
for the development of new methods for the enzymatic
synthesis of chiral cyanohydrins.
Seeds of Eriobotrya japonica were extracted in a
buffer containing polyvinylpyrrolidone for protection of
the enzyme from phenolic compounds in the crude plant
extract that form complexes by hydrogen bonding
with peptide bond oxygen or by covalent modification
of amino acid residues, causing inactivation of the
enzyme.29) The purified EjHNL had a monomer of a
single subunit. Although no chemical identification was
done, the enzyme was considered to be a glycoprotein,
since it was absorbed to a Concanavalin A Sepharose 4B
Km kcat
(mM) (sꢂ1) (mMꢂ1 sꢂ1) (%)
kcat=Km e.e.
Entry
Substrate
Configuration
1a Benzaldehyde
11.5 1700
3.5 582
1c 2-Thiophenaldehyde 35.7 1840
148
50.8
65
81
90
24
88
89
36
38
14
4
R
R
Sa
R
R
R
R
R
R
R
1b p-Anisaldehyde
161
—
1d 1-Naphthaldehyde
1e 2-Naphthaldehyde
1f Piperonal
—
—
1.1 1160
2.6 334
101
29.2
1g Propionaldehyde
1h Isobutyraldehyde
1i Pivaldehyde
99.9 223
41.8 110
39.3 164
19.4
9.6
14.3
—
1j Cyclohexane
carboxaldehyde
—
—
aThe (S)-configuration was assigned according to Cahn-Ingold-Prelog.43)
and Phlebodium aureum.23) Secondly, p-hydroxyman-
delonitrile lyase (EC 4.1.2.11) is a (S)-HNL that
catalyzes the decomposition of (S)-mandelonitrile and
(S)-4-hydroxymandelonitrile as natural substrates. p-
Hydroxymandelonitrile lyases have been investigated in
plant extracts of Baliospermum montanum,5) Sorghum
bicolor,24) Annona cherimola,20) Annona squamosa,20)
and Ximenia Americana.25) Thirdly, acetone cyanohy-
drin lyase (EC 4.1.2.37) is a (R)-HNL that catalyzes the
degradation of acetone cyanohydrin in Linum usitatissi-
mum.26) Finally, hydroxynitrilase (EC 4.1.2.39), a (S)-
HNL catalyzing the decomposition of acetone cyanohy-

1520
T. UEATRONGCHIT et al.
column, which binds specifically to the molecules
containing sugar residues. Moreover, a broad single
band of purified enzyme was observed on SDS–PAGE,
indicating that the enzyme is a typical glycoprotein.
Similar thick bands of glycoprotein on SDS–PAGE
have been reported in N-glycanase from rice seeds,30)
ꢂ-glucosidase from Schizosaccharomyces pombe,31) and
glycoprotein produced by Phoma tracheiphila.32) The
glycoprotein HNL was observed in Prunus dulcis,
Prunus laurocerasus, Prunus serotina, Prunus lyonii,
Sorghum bicolor, and Ximenia Americana,22,25,33–35)
while non-glycosylated HNLs were found in Hevea
brasiliensis, Manihot esculenta, Linum usitatissimum,
and Phlebodium aureum.23,28,29,33) HNLs isolated from
Rosaceae (Prunoideae and Maloideae subfamilies) are
glycoproteins with molecular masses of 50–80 kDa.16)
Furthermore, several isozymes of HNL from Prunus
species have been isolated, differing slightly in molecu-
lar mass, isoelectric point, or carbohydrate side chains,
etc.34) Prunus dulcis containing three or four isozymes,
Prunus laurocerasus containing three isozymes, and
Prunus serotina containing five isozymes have been
reported.33) In this study, a single purified isozyme of
EjHNL was observed according to the following criteria
on the purified enzyme: The results of column chroma-
tography showed a single symmetrical peak of purified
enzyme. Single values for molecular mass, optimum pH,
optimum temperature, and N-terminal amino acid
sequence were observed.
The absorption spectra and N-terminal sequence of
the purified EjHNL were similar to FAD-containing
HNLs. Moreover, in current work in our group on
cloning of the gene encoding EjHNL, we found a
conserved FAD-binding region in the gene encoding the
enzyme (data not shown). Therefore, EjHNL is most
likely a FAD-containing HNL. The unique characteristic
of HNLs isolated from the Prunoideae and Maloideae
subfamilies of Rosaceae is the presence of FAD. A
previous study found that the FAD is bound near the
active site.36) This prosthetic group does not play a role
in redox reactions, but it is essential for catalysis, and
it provides structural stability to the enzyme. Removal
of FAD causes inactivation of the enzyme.14,37,38)
showed optimum pH of 5.0–6.5,23,25) while the optimum
pH values for Linum usitatissimum, Manihot esculenta,
and Hevea brasiliensis HNL were around pH 5.0–6.0
with acetone cyanohydrin as a substrate.22,28,33) The
optimum temperature of the purified EjHNL was 40 ꢀC,
while the HNL obtained from Phlebodium aureum
had an optimum temperature of 35–40 ꢀC on the same
substrate.23) It was difficult to determine the exact
optimum pH and temperature of HNL from the decom-
position of the substrate due to the effect of the
spontaneous decomposition of the substrate at higher
pH and temperature. Excellent stability of EjHNL over a
wide range of pH levels (pH 3–9) and temperatures (0–
60 ꢀC) was observed. HNLs from Prunus dulcis and
Sorghum bicolor have been reported to be stable over a
wide pH range,35) while HNL from Linum usitatissimum
showed stability at pH 6–11, but was less stable under
acidic conditions.26) HNL from Hevea brasiliensis
was stable for several h at 30 ꢀC, but above 70 ꢀC was
inactivated rapidly.40) HNL from Sorghum bicolor
showed good stability against heat inactivation; the
activity remaining after incubation (60 ꢀC, 60 min) was
higher than 60%, and complete inactivation was ob-
served at 70 ꢀC (30 min).35)
Metal ions were not required for enzyme activity. The
enzymatic activity was inhibited by some heavy metal
ions that might react with essential sulfhydryl groups,
causing inactivation of the enzyme. Similar results as to
metal ions have been obtained for HNL from Ximenia
americana, Phlebodium aureum, Sorghum bicolor, and
Prunus serotina.23,25,31,41) The cysteine, serine, and
histidine amino acid residues might be located near the
active site and might be involved in the catalysis. In a
similar HNL from Prunus amygdalus, an FAD contain-
ing HNL, the identified substrate binding site and
catalytic site of the enzyme consisted of His-497, Ser-
496, Tyr-457, and Cys-328. They formed hydrogen
bonding interactions with the hydroxyl group of the
substrate. His-497 also acts as a protonating or depro-
tonating residue to the carbonyl compound and HCN in
the catalysis reaction.42) Based on the inhibition experi-
ment, it is likely that these residues are also located
around the active site of EjHNL. The occurrence or
non-occurence of these residues around the active site
of EjHNL should be made clear by our future primary
structure elucidation.
The Michaelis-Menten constant (Km) of EjHNL for
mandelonitrile was 161 mM. A lower Km value character-
izes higher affinity between substrate and enzyme.39)
HNLs from Prunus serotina (Km of 172 mM), Prunus
dulcis (Km of 290 mM), and Sorghum bicolor (Km of
790 mM) exhibited higher Km values than EjHNL, while
a Km of 93 mM was observed in Prunus lyonii.22,34)
The optimum pH for EjHNL using the natural
substrate mandelonitrile was found to be approximately
5.5. The optimum pH varies slightly for different
substrates or conditions. The optimum pH for FAD-
containing HNLs for the decomposition of mandelo-
nitrile was in the range of 5.0–7.0.22,33,34) A non-FAD-
containing HNL (Phlebodium aureum and Ximenia
americana HNL) catalyzing the same reaction also
The substrate specificity of EjHNL for the synthesis
of chiral cyanohydrins was investigated by measuring
the initial velocity of the enzymatic reaction toward the
selected aldehydes, and the configuration of the chiral
cyanohydrins was assigned according to Cahn-Ingold-
Prelog.43) Previous studies on chiral cyanohydrin syn-
thesis focused on enantiomeric excess (e.e.) and the
conversion of products, neglecting to measure the initial
velocity of the reaction, which is the basis of enzymol-
ogy.44–46) Even though several two-phase systems are
used to produce chiral cyanohydrins, a buffer system
was employed in this study to avoid the effects of

Purification and Characterization of EjHNL
1521
Industry,’’ eds. Collins, A. N., Sheldrake, G. N., and
Crosby, J., John Wiley and Sons, West Sussex, pp. 279–
299 (1992).
organic solvents on the stability and activity of the
enzyme and effects of the partitioning of substrates
between organic and aqueous phases. All aldehydes
were dissolved in DMSO to increase the solubility of the
substrates in buffer, and KCN was used as a source of
cyanide to avoid working with highly toxic HCN. The
reaction was performed at pH 4.0, at which the chemical
addition of cyanide to aldehydes was mostly suppressed.
EjHNL catalyzed the synthesis of cyanohydrins from
both aliphatic and aromatic aldehydes. It can be inferred
that EjHNL preferred aromatic to aliphatic substrates,
suggesting that the active site of the enzyme is located
near the hydrophobic region, and suggesting greater
specificity for small substrates than bulky substrates.
These results indicate potential use of EjHNL in the
synthesis of cyanohydrins. The enzyme characterized
here can be categorized as a mandelonitrile lyase (EC
4.1.2.10) because it was (R)-HNL active toward man-
delonitrile and its N-terminal sequence was similar to
that of the mandelonitrile lyases reported in this group.
HNL from Prunus dulcis, Prunus mume, Manihot
esculenta, and Hevea brasiliensis have shown broad
specificity for aliphatic and aromatic substrates,47,48)
while HNL from Linum usitatissimum had a narrow
range of substrates.26) HNL from Ximenia americana
showed fairly good specificity toward aromatic alde-
hydes.25) Specificity accepting more bulky substrates
might be improved by genetic engineering. (S)-HNLs
from Hevea brasiliensis and Manihot esculenta have
been engineered by replacing bulky amino acids, which
constrict the entrance to active sites, with less bulky
amino acids, leading to changes in the specificity of
the enzyme accepting larger substrates.47) The e.e. of
chiral cyanohydrins synthesized by EjHNL can be
further improved by optimizing the reaction conditions
to minimize non-enzymatic reactions in organic media.
It is of interest to determine the entire structure of
EjHNL and to engineer an enzyme suitable for the
synthesis of cyanohydrins.
¨
3) Wohler, F., and Liebig, J., Uber die bildung des
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acknowledged. This research was supported in part
by grants from The Japan Society for the Promotion
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