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J Sci Food Agric 1997, 75, 258È262
A Simple and Rapid Method for Isolating Cassava
Leaf Linamarase Suitable for Cassava Cyanide
Determination
Hock-Hin Yeoh,* J Howard Bradbury” and Sylvia V Egan
Division of Botany and Zoology, The Australian National University, Canberra, ACT 0200, Australia
(Received 23 July 1996 ; revised version received 5 March 1997 ; accepted 26 March 1997)
Abstract : A simple method was developed for isolating cassava leaf linamarase.
It involved homogenising the leaves in a bu†er containing polyvinylpolypyrrolidone, followed by ammonium sulphate precipitation, Ðltration and partial puriÐcation of the enzyme by hydrophobic interaction chromatography. All stages of
the enzyme preparation were carried out at room temperature and it was completed within 90 min. A linamarin-indicator strip for checking the activity of the
enzyme preparation was also developed. This isolation technique which has been
developed into a simple kit should be suitable for laboratories in developing
countries where the enzyme is needed to determine the cyanogenic potential of
cassava and its products.
J Sci Food Agric 75, 258È262 (1997)
No. of Figures : 0. No. of Tables : 2. No. of References : 21
Key words : cassava, linamarase preparation, simple kit, enzyme isolation, hydrophobic interaction chromatography, cyanide determination
existing procedures (Wood 1966 ; Cooke et al 1978 ;
Cooke 1979 ; Eksittikul and Chulavanatol 1988 ; Yeoh
1989 ; Mpkong et al 1990) or because the cost of commercial linamarase is prohibitive.
One possible solution to this problem was to develop
an acid hydrolysis method (Bradbury et al 1991) which
removed the need for enzyme. Another approach would
be to develop a simple procedure for linamarase (a bglucosidase, EC 3.2.1.21) isolation that could be used by
laboratories in developing countries, bearing in mind
that they may lack instruments for protein isolation. It
would also be useful to develop an instrument-free
method for checking the activity of the enzyme preparation. In this paper, we describe a simple method for
isolation of linamarase from cassava leaves, which was
tested on 10 di†erent varieties of cassava.
INTRODUCTION
Cassava (Manihot esculenta Crantz) is widely grown for
its edible storage roots (Cock 1985). However, the presence of cyanogenic glucosides, mainly linamarin, and
their breakdown products in cassava and its processed
products, has been a cause of concern from the viewpoint of food safety. Many methods for measuring total
cyanide content are available (Cooke 1978 ; Bradbury et
al 1991 ; Bradbury and Egan 1992 ; Yeoh and Truong
1993 ; Yeoh 1993 ; Bradbury et al 1994 ; Yeoh and Tan
1994a,b ; Brimer 1994 ; Tatsuma et al 1996 ; Yeoh et al
1996). Several of these methods are based on the enzymatic hydrolysis of linamarin, followed by spectrophotometric,
potentiometric
or
amperometric
measurement of the cyanide released. Unfortunately,
enzyme-based methods cannot be used in many laboratories in developing countries, either because of lack of
the equipment needed for enzyme preparation using
EXPERIMENTAL
Plant material
* On sabbatical leave from School of Biological Sciences, The
National University of Singapore, Kent Ridge, Singapore
119260.
” To whom correspondence should be addressed.
Leaves of cassava plants were obtained from a collection maintained in the Plant Culture Facility at the
Australian National University, Canberra.
258
( 1997 SCI.
J Sci Food Agric 0022-5142/97/$17.50.
Printed in Great Britain
Isolation of cassava linamarase
Enzyme preparation
About 2 g of fresh, fully expanded young leaves were
used. They were homogenised in a mortar in 10 ml
100 mM Na citrate at pH 6 containing 10 g litre~1 polyvinylpolypyrrolidone, followed by the addition of 12 ml
of 2 M (NH ) SO . The e†ective molarity of the crude
42 4
extract was about 1 M (NH ) SO . The homogenate
42 4
was allowed to stand for 30 min at room temperature,
then Ðltered through a layer of Ñour-sack cloth on a
Ðlter funnel. The Ðltrate became clear (slightly tinted
yellow) after 10È12 ml of Ðltrate (green) had passed
through. At this point, collection of the clear Ðltrate was
continued in a measuring cylinder. The initial collection
of Ðltrate (green) was gently poured back on the Ñoursack cloth Ðlter. Alternatively, Ðltration through
Whatman No 1 Ðlter paper gave a clear Ðltrate and was
quite satisfactory. About 10È13 ml of the clear Ðltrate
was recovered.
A phenyl Sepharose CL-4B (Pharmacia Biotech,
Sweden) column was prepared by using a 5 ml syringe
barrel which was Ðlled up to the 2 ml mark with the
phenyl Sepharose slurry. This was washed with 20 ml
water and then equilibrated with 10 ml 1 M (NH ) SO
42 4
in 50 mM Na citrate at pH 6. The clear Ðltrate (10È
13 ml) was then passed through the equilibrated phenyl
Sepharose column followed by 6 ml 0É3 M (NH ) SO
42 4
in 50 mM Na citrate at pH 6 then 6 ml water. The
eluate from the water wash was collected and contained
the linamarase. The phenyl Sepharose CL-4B could be
regenerated by soaking in 0É1 M NaOH for about
30 min, then washing it with water until the washings
were neutral.
For some cassava varieties, we found it better to use
12 ml 0É8 M (NH ) SO instead of 2 M (NH ) SO in
42 4
42 4
the enzyme extraction step (see above) and in this case
the phenyl Sepharose CL-4B was equilibrated with
0É4 M (NH ) SO (instead of 1 M (NH ) SO , see
42 4
42 4
above) in 50 mM Na citrate at pH 6. The bound proteins were desorbed as before.
Protein determination
This was carried out using the Bio-Rad protein assay
kit (Bradford, 1976).
Enzyme assay
For convenience, we monitored the b-glucosidase activity and for the Ðnal preparation we also determined the
linamarase activity. One unit (U) of enzyme activity was
deÐned as 1 kmol of the substrate hydrolysed in 1 min.
The b-glucosidase assay was performed using 5 mM pnitrophenyl-b-D-glucoside in 50 mM Na citrate at pH 6
(Yeoh 1989). The total reaction volume was 600 kl.
After incubation at 37¡C for 15 min, 3 ml 100 mM
Na CO was added to the reaction mixture and the
2
3
259
absorbance measured at 420 nm. Linamarase activity
was determined using 5 mM linamarin in 50 mM Na
citrate at pH 6 at 37¡C for 15 min. The total reaction
volume was kept at 600 kl (Yeoh 1989). The glucose released was determined using the glucose oxidase method
(Raabo and Terkildsen 1960).
Preparation of linamarin-indicator paper strip
An aliquot (20 kl) of 20 mM linamarin was spotted on a
piece of 10 ] 15 mm Whatman chromatography paper
grade 17 Chr and left to dry at room temperature.
Whatman 3MM chromatography paper (10 ] 15 mm)
soaked in alkaline picrate solution (Williams and
Edwards) 1980) was also left to dry at room temperature. They were then glued onto a 10 ] 40 mm
strip of plastic cut from an overhead transparency sheet.
The linamarin-paper was placed at one end of the
plastic strip and the picrate paper placed next to it, with
a 2È3 mm gap between them. A water-based commercial glue was used for this purpose. To test the
enzyme activity 50È100 kl of the enzyme preparation
was spotted on the linamarin-paper. It was immediately
placed in a clear plastic bag designed to Ðt 10 ] 40 mm
paper strips and the bag sealed with tape. Alternatively,
an air-tight bottle measuring about 14 mm id ] 75 mm
could be used. It was incubated at 37¡C, or at 30¡C. If
the picrate paper changed from yellow to brick red in
15 to 30 min, then the enzyme preparation had high linamarase (b-glucosidase) activity. The reaction was
observed to be faster at 37¡C than at 30¡C. If the picrate
paper changed from yellow to brown or brick-red overnight, the enzyme preparation was low in activity but
might still be useful for cassava cyanide determination.
RESULTS AND DISCUSSION
Several methods for isolating cassava linamarase are
available (Wood 1966 ; Cooke et al 1978 ; Cooke 1979 ;
Eksittikul and Chulavanatol 1988 ; Yeoh 1989 ; Mpkong
et al 1990), but many laboratories in developing countries are unable to prepare the enzyme because they
lack the facilities to carry out these procedures. Even in
a simple isolation method which involves only ammonium sulfate precipitation (Cooke 1979), a centrifuge was
needed. The method developed here eliminates the use
of a centrifuge.
Separation of leaf debris and other precipitated proteins was achieved by simple Ðltration. We found that
Ñour-sack cloth which is available in the tropics, was
suitable for this purpose, although Whatman No 1 Ðlter
paper was satisfactory. Instead of using ammonium sulphate to precipitate the linamarase as employed by
most methods, we used this salt to facilitate the hydrophobic attachment of the enzyme present in the soluble
form to the phenyl Sepharose CL-4B. The bound proteins were desorbed from the column using water and
H-H Y eoh, J H Bradbury, S V Egan
260
the simple batch elution procedure did not require any
instruments associated with column chromatography.
Overall, the steps were simple to follow and it took
about 90 min to prepare the enzyme. It was found possible to scale up the isolation protocol using 10 g
cassava leaves instead of 2 g. However, the Ðltration
step and binding of the proteins to the phenyl Sepharose column took a longer time, which increased the
total time needed to prepare the enzyme.
Leaves of 10 cassava varieties were used to assess if
the method would be useful for cassava in general, as
many di†erent varieties are grown in the tropics. In this
protocol, the homogenate was brought to about 1 M
(NH ) SO and the proteins adsorbed to phenyl Sep42 4
harose CL-4B in 1 M (NH ) SO (Table 1). Under these
42 4
conditions, the preparations showed enzyme purities
ranging from about 3È9-fold and recoveries of 30È81%,
depending on the cassava variety. Preparations from
the Ðrst six entries in Table 1, gave high b-glucosidase
activity (2É4È14É5 U) and they exhibited high linamarase
activities (118È680 U mg~1 ; see Table 1). On the other
hand, the last four entries in Table 1 gave much lower
enzyme activities ; the total b-glucosidase activity in
the crude enzyme Ðltrate was only 0É13 to 0É8 U (see
Table 1).
In an attempt to overcome this problem of low
amounts of enzyme being extracted, we modiÐed the
extraction conditions. Changes were made to the
amount of polyvinylpolypyrrolidone and/or (NH ) SO
42 4
used. Using variety TMS 60506, it was observed that by
increasing the polyvinylpolypyrrolidone to 20 g litre~1
and retaining the e†ective molarity of (NH ) SO at
42 4
1 M, the enzyme activity in the crude preparation was
still low (total b-glucosidase activity, 0É13 U ; see Table
2). On the other hand, when the e†ective molarity of
(NH ) SO was reduced from 1É0 to 0É4 M and the poly42 4
vinylpolypyrrolidone content retained at 10 g litre~1,
the crude enzyme Ðltrate showed an increase of nearly
100 fold in enzyme activity (total b-glucosidase activity,
12É7 U). Correspondingly, the Ðnal preparation was
higher in linamarase activity (382 U mg~1 protein)
compared to results of earlier extraction procedures (see
Table 1). It seems likely that with cv TMS 60506 some
of the linamarase was precipitated along with other proteins and lost in 1É0 M (NH ) SO , but not in 0É4 M
42 4
(NH ) SO . Increasing polyvinylpolypyrrolidone to
42 4
40 g litre~1 and retaining the use of 0É4 M (NH ) SO ,
42 4
however, decreased the level of extractable enzyme
activity (total b-glucosidase activity, 1É8 U). Based on
these data, we used a lower (NH ) SO concentration
42 4
to prepare the enzyme from TMS 30572, TMS 71693
and MCOL 1468 (Table 2). The results showed that
only TMS 30572 gave a high enzyme activity (total bglucosidase activity in crude extract, 10É3 U ; linamarase
activity of Ðnal preparation, 528 U mg~1 protein) while
the other two varieties still gave low enzyme activities in
their preparation (total b-glucosidase activity, 0É2È
0É3 U). The di†erences observed between the 10 cassava
cultivars studied showed that di†erent extraction conditions were necessary to obtain the best results for di†erent varieties.
Because of the variability of the linamarase activity
obtained from di†erent cassava cultivars, it was important to check the level of enzyme activity in the preparation.
In
this
respect,
we
developed
a
linamarin-indicator paper strip in which about 50 kl
TABLE 1
Preparation of cassava leaf linamarase (b-glucosidase) from cassava varieties
Cassava
variety
Crude enzyme Ðltrate
T otal
protein
(mg)
SM1 150
M AUS 7
TMS 4(2) 1425
TMS 91934
TMS 63397
TMS 50395
TMS 71693
TMS 30572
TMS 60506
MCOL 1468
3É5
2É4
1É3
1É9
0É7
0É9
0É6
0É6
0É4
0É5
b-Glucosidase
activity
T otal
activity
(U)
SpeciÐc
activity
(U mg~1)
19É5
10É9
10É6
11É5
3É2
6É1
0É8
0É4
0É13
0É13
5É6
4É5
8É2
6É1
4É6
6É8
1É3
0É7
0É3
0É3
Preparation from Phenyl Sepharose CL -4B
T otal
protein
(mg)
0É7
0É6
0É2
0É4
0É1
0É1
0É05
0É05
0É04
0É02
b-Glucosidase
activity
T otal
activity
(U)
SpeciÐc
activity
(U mg~1)
14É5
8É6
8É6
9É1
2É4
4É6
0É6
0É17
0É06
0É04
20É7
14É3
43É0
22É8
24É0
46É0
12É0
3É4
1É5
2É0
Puritya
(Fold)
Recoverya
(%)
3É7
3É2
5É2
3É7
5É2
6É8
9É2
4É9
5É0
6É7
74
79
81
79
75
75
75
50
46
30
L inamarase
activity
(U mg~1)b
320
118
495
348
530
680
159
26É4
1É8
10É8
(37É3)
(11É7)
(19É0)
(21É0)
(6É0)
(13É0)
(1É4)
(0É2)
(0É01)
(0É04)
a Purity and recovery were based on b-glucosidase activity, purity \ col 7/col 4, recovery (%) \ col 6/col 3 ] 100.
b Values in parentheses refer to U ml~1 of Ðnal enzyme preparation.
Isolation of cassava linamarase
261
TABLE 2
Preparation of cassava leaf linamarase (b-glucosidase) following some modiÐcations of the extraction conditions
Cassava
Crude enzyme Ðltrate
T otal
protein
(mg)
TMS 60506c
TMS 60506d
TMS 60506e
TMS 30572d
TMS 71693d
MCOL 1468d
a
b
c
d
e
0É4
1É3
0É6
0É8
0É8
0É4
b-Glucosidase
activity
T otal
activity
(U)
SpeciÐc
activity
(U mg~1)
0É13
12É7
1É8
10É3
0É3
0É2
0É3
9É8
3É0
12É9
0É4
0É5
Preparation from Phenyl Sepharose CL -4B
T otal
protein
(mg)
0É04
0É3
0É07
0É14
0É04
0É04
b-Glucosidase
activity
T otal
activity
(U)
SpeciÐc
activity
(U mg~1)
0É08
9É3
1É4
7É6
0É06
0É04
2É0
31É0
20É0
76É0
1É5
1É0
Puritya
(Fold)
Recoverya
(%)
6É7
3É2
6É7
5É9
3É8
2É0
80
73
78
74
20
20
L inamarase
activity
(U mg~1)b
19É2
382
208
528
3É6
1É2
(0É1)
(19É1)
(3É5)
(8É8)
(0É02)
(0É01)
Purity and recovery were based on b-glucosidase activity, purity \ col 7/col 4, recovery (%) \ col 6/col 3 ] 100.
Values in parentheses refer to U ml~1 of Ðnal enzyme preparation.
Extraction medium contained 20 g litre~1 polyvinylpolypyrrolidone, followed by addition of 2 M (NH ) SO .
42 4
Extraction medium contained 10 g litre~1 polyvinylpolypyrrolidone, followed by addition of 0É8 M (NH ) SO .
42 4
Extraction medium contained 40 g litre~1 polyvinylpolypyrrolidone, followed by addition of 0É8 M (NH ) SO .
42 4
(one drop) of the enzyme preparation was added to the
linamarin on the paper. Using the enzymes prepared in
Table 1, it was observed that preparations with linamarase activity greater than 6 U ml~1 changed the
picrate paper from yellow to brick-red within 15 min at
37¡C. Enzyme preparations from the four varieties that
gave low linamarase activity required an overnight
incubation (about 20 h) to turn the picrate paper
drown.
We also compared this response with their ability to
hydrolyse the linamarin in cassava Ñour. Here, we
added 50 kl of the enzyme preparation to 100 mg
cassava Ñour in 450 kl phosphate bu†er at pH 8 in an
air-tight bottle in which a piece of picrate paper was
suspended. After incubation at 30¡C for about 20 h, the
colour of the picrate paper was compared against the
control which contained sufficient linamarase to hydrolyse the linamarin in the Ñour sample. It was observed
that the six enzyme preparations with high linamarase
activity were able to hydrolyse completely the linamarin
in the Ñour. For preparations with low enzyme activity,
namely those from varieties TMS 60506, TMS 71693,
TMS 30572 and MCOL 1468, 200 kl of the enzyme
preparation was required to give the same result.
Although it was possible to use enzyme preparations
with low linamarase activity, we would suggest using
those with high enzyme activity on the basis of productivity, since a 6 ml preparation from 2 g leaves could
be used for about 120 analyses, assuming 50 kl were
needed for each analysis.
Overall, a simple and rapid method of preparing
cassava leaf linamarase was developed. The linamarinindicator paper could be used to check the activity of
the enzyme preparation and from its response one could
use a suitable amount for the determination of linamarin or cyanogenic potential in cassava. Since the procedure does not require any equipment such as a
centrifuge or fraction collector, it should be suitable for
laboratories in developing countries. This should enable
researchers and food analysts to use the enzyme-based
methods of linamarin and cyanogenic potential determination, thus making safer the consumption of cassava
and cassava products. A simple kit has been developed
for the preparation of linamarase from cassava leaves
using this method.
ACKNOWLEDGEMENTS
HHY is a Visiting Fellow at the Australian National
University on study leave from the National University
of Singapore. The authors thank Mr Chew Boon Swee
of Exclusive Mark (M) Sdn Bhd, Malaysia for kindly
donating the water-base commercial glue. Thanks also
to the Australian Centre for International Agricultural
Research (ACIAR) for Ðnancial support of this work.
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