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Isolation, Partial Purification and Application of lectin isolated from

Phaseolus vulgaris (Red Kidney Bean)

Pallavi Dongre 1, Nagma Shaikh 1, Pragati Gangwal 1, Kalyani Pawar*

PG Student 1, Assistant Professor*, Department of Biotechnology, Shivchhatrapati College,
Aurangabad.

Received: February 09, 2019 Accepted: March 29, 2019

ABSTRACT: : The lectin contents in some parts of plants are higher. Lectins are found in abundance in
legume seeds. Phaseolus vulgaris is an herbaceous annual plant grown worldwide for its edible beans. In
the present study, lectin has been isolated from seeds of Phaseolus vulgaris. Isolated lectin was partially
purified by using preliminary fractionation i.e. by ammonium sulphate precipitation method, at 30%, 60%,
70%, and 90% saturation respectively followed by dialysis. The protein concentration was determined by
using Lowry method for all the fractions of lectin’s. More specifically the study aimed to determine the
lectin’s blood group specificity and pH stability. The isolated lectins show remarkable hemagglutination
activity. The hemagglutinating activity of isolated lectin was found to be stable in the temperature range 0
°to 70°C and the pH range 4-9. Tests of the inhibition of hemagglutinating activity by various
carbohydrates were performed. We investigated antibacterial activities of isolated lectin by agar well
diffusion technique, on various strains of bacteria.

Key Words: Phaseolus vulgaris, ammonium sulphate precipitation, hemagglutination, antibacterial.

Introduction
Lectins are defined as proteins /glycoproteins with at least one non catalytic domain which bind reversibly
to a specific mono or oligosaccharides. The lectin contents in some parts of plants are higher, e.g.,390 & 75
mg of the purified lectin was recovered from 100mg Remusatia vivipara tubers (Bhat et al. 2010) and
Astragalus mongholicus roots ( Yan et al. 2005), respectively. Lectins are also found in seed. The lectin
content in non legume plants is low, e.g., 3.3 mg lectin from 100 g Hibiscus mutabilis seeds (Lam and Ng
2009). Lectins are found in abundance in legume seeds. Phaseolus vulgaris is an herbaceous annual plant
grown worldwide for its edible beans, popular in both dry & green bean forms. The commercial production
of beans is well distributed worldwide. There are different varieties, including anasazi beans, black beans,
cranberry bean, borlotti beans, pink beans, pinto beans, kidney beans, shell beans, white beans, yellow
beans & French beans, etc. Lectins or hemagglutinins have been purified from different varieties of P.
vulgaris. Plants lectin’s are the first & the most extensively studied lectins due to their broad distribution.
(Lirner, I.E., Sharon, N.,& Goldstein, I. J.1986). Since the last century, numerous plant lectins have been
purified & their bioactivities & sugar binding specificities have been characterized.
(Liu,B.,Bian,H.J.,&Bao,J.K.2010). The majority of plant lectins do not exhibit blood group specificity, but some
of them display species specificity in hemagglutinating activity. Others have relatively universal activity
towards red blood cells. Plant lectins serve as tools to distinguish between malignant and benign tumors
and determine the metastatic status by their degree of glycosylation. A few plant lectin manifested
antifungal activity. It is believed that lectins do not directly inhibit fungal growth. Some plant lectins are
resistant to heat, heat processing can reduce their toxicity. In the present study, lectins is isolated from
Phaseolus vulgaris, which shows significant antibacterial activity against S. aureus.

Materials and method:

I. Collection: The Red kidney bean seeds were collected for isolation and partial purification of
lectin from a local food store situated at Aurangabad.
II. Isolation of Phaseolus vulgaris lectin: Extraction of Phaseolus vulgaris lectin was carried out
according to process by Zhang et al. Phaseolus seeds (25 g) were ground to a powder and 250
ml of 50 mM phosphate buffer (pH 7.2) were added. Then, the homogenized powder in buffer
was left over night for complete extraction at 4˚c. After filtration, the filtrate was centrifuged at
8000 rpm for 20-25 min and the supernatant was fractionally precipitated with ammonium
sulfate at 30% ,60% 70% and 90% saturation, respectively. The pellets were collected in
separate tubes each containing 15 ml phosphate buffers (pH 7) [10].
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III. Partial purification of Phaseolus vulgaris lectin: When the ionic strength of a protein
solution is increased by adding salt, the solubility decreases, and protein precipitates. The salt
molecules compete with the protein molecule in binding with water. The concentration of salt
requires for precipitation of the protein out of the solutions is varies greatly in different
proteins. It also used to concentrate dilute solution of proteins. Ammonium sulphate salt was
taken in the salting out process. According to the salt chart ammonium sulphate were added to
the crude by pinch wise and continues stirring was done by magnetic stirrer. Then the sample
was stored for overnight at 4˚c and in the next day the sample was taken for centrifuge, then
supernatant and pellet was collected. The supernatant was taken and ammonium sulphate salt
was added in pinch wise and continuous stirring was done by magnetic stirrer. Similarly
supernatant was collected and measured ammonium salt was added for dialysis and stored at
4˚c overnight. The pellet was collected after centrifuge and undergo dialysis in PBS for 3-4 days
[2].
IV. Determination of concentration of protein: The protein concentration of lectin was
determined by using Lowry Method using bovine serum albumin as the standard protein [2].
V. Assay of hemagglutinating activity: The hemagglutination activity was performed initially
using normal human erythrocytes (A). Fresh erythrocyte were separated from plasma by
centrifugation at 3000 rpm for 4 minutes at 5-10 ˚c and washed extensively with phosphate
buffer saline (PBS). Finally, 3% suspension of erythrocytes was prepared in PBS and
hemagglutination tests were performed in standard microtitre plate by the two-fold serial
dilution method (Liener et al, 1962). A 50µl aliquot of the erythrocyte suspension was mixed
with 50µl of serially diluted lectin. Agglutination assay was examined visually after incubation
for 1 hrs at room temperature .The unit of hemagglutination activity (U) termed as titer was
expressed as the highest dilution of the lectin that showed complete agglutination. [1]
VI. Sugar specificity: The sugar specificity investigates inhibition of lectin induced
hemagglutination by various carbohydrates were performed in a manner analogous to the
hemagglutination test. 10, 50,100 mM of each sugar sample was prepared in phosphate buffer
saline .All of the sugar sample was mixed with an equal volume (25µl) of a solution of the lectin.
The mixture was allowed to stand for 30 min at room temperature and then mixed with 50µl of
a 2% human erythrocyte suspension .The known sugar which gives an agglutination with blood
suspension and no precipitate of red blood cell that means the tested lectin specific to that
sugar type.[5]
VII. Effect of pH on isolated lectin induced Hemagglutination: The effect of pH was examined by
preparing lectin solution with pH 4 to 9 using the following reagents: pH 4&5 (citrate acetate
buffer), pH 6-7(Phosphate buffer), pH 8-9 (Tris HCl). Equal volumes of lectin sample and pH
solution were mixed and incubated at room temperature for 30 min. The hemagglutination
assay was then performed. Absorbance was measured at 450nm.[4]
VIII. Effect of Temperature on isolated lectin induced hemagglutination :
The effect of temperature was examined by diluting the sample 2 fold and heating at 20℃ to
80⁰ for 30 min. The sample was allowed to cool down to room temperature. The
abovementioned hemagglutination assay was performed on these samples. Absorbance was
measured at 450nm. [4].
IX. Antibacterial activity : The antibacterial activity of partially purified lectin
(30%,60%,70%,90%) solution against the bacterial pathogen including E. coli, S. aureus,
Pseudomonas, Klebsiella, S. typhi was evaluated by using agar well diffusion technique. Broth
culture of the above mentioned bacterial pathogens compared to Mac Farland’s standard 0.5
were prepared. Lawn culture of these bacterial pathogens were made on the Muller Hinton agar
plates & the plates were dried for 15 min. Wells measuring 6mm in depth was made on the agar
using sterile cork borer . 100µl of partially purified lectin (30%,60%,70%,90%) solutions was
added to the wells. 100µl of 5Mm phosphate buffer solution (pH7.2) was used as a control. The
plates were incubated at 37℃ overnight & the zone of inhibition of growth was measured in
mm. The entire test was done in triplicates. [10]

Results:
Lectins were isolated from red kidney beans, which was partially purified by using ammonium
sulfate precipitation method (30%,60%,70%,90%) as shown in fig. A and B.

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Fig1 (A) Ammonium sulfate precipitation & (B) Dialysis

Determination of concentration of protein :- The protein concentration was determined for each
% of isolated lectin which was found to be 29 µg/ml for 30% ,40 µg/ml for 60%, 40 µg/ml for 70% &
34 µg/ml for 90 % respectively.
Assay of hemagglutinating activity: - Hemagglutination activity of partially purified lectins extracted
from Phaseolus vulgaris on human blood group sample (A) was performed.
Highest hemagglutination activity titer was found to be 1.4 for all isolated lectins as shown in table 1
and in fig. C
Lectin % Human Blood Group (A)
Saturation

Hem agglutination Activity (Titer)

30% 0.795 1.080 1.00 0.593 1.453 0.982

60% 0.595 0.982 1.040 1.209 1.324 1.410

70% 0.247 0.598 1.458 1.093 1.028 1.56

90% 1.118 1.427 0.904 0.902 0.943 0.698

Table 1: Hemagglutination activity

Fig C. Hemagglutination activity

Sugar specificity:- The hemagglutinating activity of purified red kidney beans lectin could not be
inhibited by many of the simple sugars tested at 10mM, 50mM, 100mM of D(+) glucose, D(+) galactose,
D(+) fructose, D(+)sucrose D(+) xylose, D(+)maltose, D(+)dextrose ,but the hemagglutinating activity
inhibited with D(+) xylose, D(+) maltose as shown in table 2 and fig. D, E,F and G.
+: Positive Hemagglutination Inhibition, - : Negative Hemagglutination Inhibition

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Lectin fraction Hemagglutination Inhibition

Sugars
Glucose Dextrose Maltose Galactose Xylose Sucrose

30% ++ ++ + ++ - ++
60% ++ ++ + ++ - ++
70% ++ ++ + ++ - ++
90% ++ ++ + ++ - ++
Table 2: Sugar Specificity
Sugar specificity of 30% , 60% ,70% and 90% Partially purified Lectin:-

Fig. D Fig. E

Fig.F Fig. G

Effect of pH on isolated lectin induced Hemagglutination: The isolated lectin exhibited remarkable
stability over the range of pH 4-9 and loss the activity at pH 9. Hemagglutination activity at different pH
is shown in fig. H and table 3. The graph of hemagglutination activity of lectin at different pH is shown in
Graph 1.
pH Absorbance at 450nm

Control 0

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4 0.322

5 0.4

6 0.3

7 0.8

8 0.7

9 0.07

Table3: Effect of pH on isolated lectin induced Hemagglutination

Graph 1 : Effect of pH on isolated lectin induced Hemagglutination.

Fig H: Effect of pH on hemagglutination activity of Phaseolus vulgaris lectin

Effect of Temperature on isolated lectin induced hemagglutination: Highest hem agglutinating

activity of lectin observed at 40˚C .Considerable loss in activity of lectin occurred at 70°C as shown in fig.
I, table 4 and graph 2.
Temperature Absorbance at 450nm

20 1.038

30 1.079

40 1.611

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50 1.035

60 1.067

70 0.609
80 0.321
Table 4: Effect of Temperature on isolated lectin induced hemagglutination

Graph 2 : Effect of temperature on hemagglutination activity of Phaseolus vulgaris lectin

Fig I: Effect of temperature on hemagglutination activity of Phaseolus vulgaris lectin

Antibacterial activity: The antibacterial activity of partially purified lectin ( 30% , 60% ,70% 90%) is
screened against the E. coli, S. aureus, Pseudomonas, Klebsiella, S. typhi using agar well diffusion
technique and the zone of inhibition was measured in diameter which is shown in table 5 . The area of
inhibition where the growth of organism inhibited by partially purified lectin was observed to be
significant with S. aureus when compared with control followed by Pseudomonas, E. coli, Klebsiella .
Zone of inhibition by isolated lectin against different pathogens is shown in fig.J

Fig J. Antibacterial Activity

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Lectin fraction Organisms Zone of inhibion

(% Saturation) ( mm)
30% E. coli 10mm
S. aureus 19mm
Pseudomonas 13mm
Klebsiella 9mm
S.typhi -
60% E. coli 14mm

S. aureus 21mm
Pseudomonas 15mm
Klebsiella 11mm
S.typhi -
70% E. coli 12mm
S. aureus 20mm
Pseudomonas 13mm
Klebsiella 10mm
S.typhi -
90% E. coli 12mm
S. aureus 24mm
Pseudomonas 12mm
Klebsiella 12mm
S.typhi -
Table5. Antibacterial activity

Discussion:
The remarkable ability of phaseolus vulgaris lectins is that it shows hemagglutination activity [13,12].The
present study demonstrates that isolated lectin shows stable hemagglutination activity at 40 ℃ and
considerable loss in its activity was observed at 70℃. Earlier studies presented that [13].There was varying
degree of hemagglutination at the sugar concentration on all the isolated lectin in this study .These findings
suggested that all the sugar (Glucose, Dextrose, Galactose, Sucrose) did not inhibit the activity of the lectin
found in the phaseolus vulgaris. Our findings are similar to the results shown by Patricia M.G.Paiva1 etal.
[16]. Sugar including xylose and maltose did not show hemagglutination activity .This finding contradicted
results reported by [13]. In this study, isolated lectin showed antibacterial activity against E.coli,
Pseudomonas, Klebsiella, S.aureus. While lectin was devoid of antibacterial activity against S.typhi [16].

Conclusion:
In this present work, lectins were isolated from Phaseolus vulgaris which were partially purified by using
ammonium sulfate precipitation method to obtain 30%, 60%,70%, and 90% saturation respectively. Highest
protein concentration was found to be 40µg/ml in 60% and 70% of partially purified lectin.
Hemagglutination assay was performed for isolated lectin. Effect of pH, Temperature, was studied on
isolated lectin fractions. It shows that isolated lectins showed highest hemagglutination in 7 pH and shows
highest hemagglutinatinating activity at temperature 40℃.Sugar specificity of isolated lectin has been
checked which shows that the isolated lectin is not specific for Xylose. i.e. hem agglutinating activity of lectin
was inhibited by xylose. In this study , the ability of lectins from Phaseolus vulgaris to inhibit the growth of
selected microorganisms could justify their potential ability in controlling bacteria capable of causing
diseases in man and animals.

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[ VOLUME 6 I ISSUE 2 I APRIL– JUNE 2019] E ISSN 2348 –1269, PRINT ISSN 2349-5138
Acknowledgment: We are grateful to Dr. P. V. Ashtekar, Principal of Shivchhatrapati College, Aurangabad,
for providing us infrastructure and chemical for our PG project work.
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