Partial Purification and Characterization

of a lectin from Aspergillus niger

1
INTRODUCTION

 Lectins are glycoproteins or oligomeric proteins with one or more sugar

binding sites per subunit

 First designated as ‘heamaggluinins’ or ‘phytohaemagglutinins’.

 Non-immune origin, bind reversibly with specific sugars.

 Lectin recognition occurs as a result of lock and key complementarity.

 Molecular markers of cell differention.

 Carriers of metabolic inhibitors and toxic drugs.

 Bioadhesive molecules.

 Aggregation of tumor cells is mediated by lectins present on their surface.

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 OBJECTIVES

– Determination of carbohydrate specificity of A. niger lectin.

– Partial purification of lectin produced by A. niger.

– Characterization of the partially purified A. niger lectin.

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REVIEW OF LITERATURE
 Microbial lectins:
 Bacterial lectins:

– Pathogenicity

– Bradyrhizobium japonicum (Ho, 1992) – galactose specific

lectin.

– Aeromonas sobria (Hokama and Iwanaga, 1991), and

Aeromonas hydrophilla (Hokama and Nakason, 1990), and
Agrobacterium tumefacians (Depierreuox et al.,1991).

– Mannose specific lectins have been reported in

Lactobacillus fermentum, L. fermentum sub sp. cellobiosus
and L. animalis. (Gusilis et al., 1999).

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– Fungal lectins:

 Molds
– Aspergillus fumigatus (Tronchin et al., 2002).
– A. niger, A. versicolor, A. rugulosus and A. nidulans (Singh et al., 2008).
– Ganoderma lucidum (Thakur et al., 2007).
– Cordyceps militaris (Jung et al., 2007).
– Xylaria hypoxylon (Wang et al., 2006).
– Armillaria luteo-virens (Feng et al., 2006).
– Xercomus chrysenteron (Trigueros et al., 2003).

 Yeast lectins
– Kluyveromyces bulgaricus (Al-Mehmood et al., 1992).
– Sacchromyces cerevisiae (Stratford and Pearson, 1992).

– Viral lectins:
 Influenza viruses (Wiley and Skehel, 1987).

– Algal lectins:
 Ulva pertusa (Wang et al., 2004).
 Hypnea musciformis (Nango et al., 2005).
 Bryothamnion triquetrum and B. seaforthii (Ainonz et al., 1995).

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 Purification and characterization of lectins:

– Ammonium sulphate precipitation (Salting out).

– Chromatography.

 Sclerotium rolfsii – ammonium sulphate precipitation and gel-filtration.

 Pleurotus cornucopiae - mucin-sepharose affinity chromatography.

 Aspergillus fumigatus - gel filtration followed by ion-exchange

chromatography.

 Armillaria luteo-virens - ammonium sulphate precipitation, ion-

exchange chromatography and gel filtration.

 Rhizopus stolonifer - affinity charmatography.

 Codium giraffe - ammonium sulphate precipitation, affinity

chromatography and anion exchange chromatography.

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 Applications:

– identifying microorganisms
– activation of lymphocytes
– indication of synthesis of specific proteins and
enzymes
– separation of cells
– mitogens for T-cells
– targetting vaccines to the epithilium and association
of vaccines with biodegradable particles
– insecticidal properties
– toxic effects on pests

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MATERIALS AND METHODS

 Procurement and maintenance of

microbe:
Aspergillus niger maintained on Potato Dextrose Agar
(PDA) slants.

Composition :

Potato starch - 20.0% (w/v)

Dextrose - 2.0% (w/v)
Agar - 3.5% (w/v)
pH - 6.0

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  Extraction of lectin

Fungal biomass obtained by filtering the broth

Washed with distilled water, 0.1M phosphate buffered saline & pressed dry

Recovered mycelium homogenized in PBS in ratio 1:2 at speed for 3-5 min in an ice bath

Extract centrifuged at 300 x g for 2 min at 4 °C

Supernatant assayed for lectin activity

 Collection of blood:
Blood samples from human volunteers and animals were drawn in Alsever’s
solution in the ratio 1:2 and stored at 4 °C for use following 4-5 days.

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  Preparation of cell suspension:
– Preparation of erythocyte suspension

Collected blood centrifuged at 400 x g, 15 min at 4 °C

Supernatant removed, pellet washed thrice with excess of PBS

Obtained erythrocytes resuspended in PBS to from 2% suspension (v/v), stored at 4 °C

– Enzymatic treatment of erythrocytes

1 ml of 10% erythrocytes +

10 ml of neurominidase (0.2 IU/ml) or

1.0 ml of protease (2 mg/ml)

Incubated at 37 °C for 60 min

Reaction stopped by adding excess of PBS

Centrifuged at 1200 rpm for 5 min

Washed 5 times with PBS

Resuspended pellet in PBS to form 2% RBC suspension 10

 – Preparation of lymphocyte suspension

Heparinized blood was mixed an equal volume of normal saline

Layered carefully onto histopaque (3:1)

Centrifuged at 1200 rpm for 20 min

An opaque ring of lymphocytes formed at interface of plasma and histopaque was

aspirated carefully

Contaminating erythrocytes were lysed using 0.82% NH4CL at 37 °C for 5 min

Washed thrice in PBS

Resuspended in PBS to a concentration of 5 x 106 cells/ml

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– Preparation of splenocyte suspension

Excised spleen of mice was teased in MEM with the help of glass slides

Centrifuged at 1200 rpm for 20 min

Pellet obtained was treated with 0.82% NH4CL at 37 °C for 5 min

Washed thrice in PBS

Resuspended in PBS to a concentration of 5 x 106 cells/ml

– Preparation of microbial cell suspension

24 hr old culture of E.coli and Kluyveromyces marxianus

Centrifuged at 5000 rpm for 20 min at 4 °C

Washed the pellet in PBS (5 times) to remove traces of media

Cells resuspended in PBS to a concentration of 1 x 106 cells/ml

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 Agglutination assay:

20 µl of two fold serial diluted lectin extract

+ equal
volume of cells suspension in wells of U-bottom microtitre plates

Plates incubated at RT for 30 min

Then incubated at 4 °C for 1-2 h

 Positive reaction – mat formation

 Negative reaction – button formation

 Lectin titre – defined as inverse of highest dilution capable of

agglutination

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  Carbohydrate inhibition assay:

20 µl of app. diluted lectin (twice the lowest concentration capable of visible agglutination)
+
equal volume of sugar
solution to be tested
Incubated for 1 h at root temperature

40 µl of 2% erythrocyte suspension (v/v) added to each well

Incubated for 30 min at room temperature

Stabilized at 4 °C for 2-3 h

 Positive control - 20 µl of PBS instead of lectin extract.

 Negative control - 20 µl of PBS instead of sugar extract

 Minimum inhibitory concentration - defined as the lowest

concentration of sugar capable of complete inhibition of agglutination.
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 Lectin activity as a function of growth:
Erlenmeyer’s flasks containing 50 ml medium

Inoculated with 5 mm culture discs

Incubated at 30 °C, stationary conditions

Lectin activity determined in cultures incubated for 3-11 days at 24 h intervals

 Partial purification of lectin:

– Ammonium sulphate precipitation

30-90% (w/v) ammonium sulphate added to 10 ml aliquots of mycelial extract with

constant stirring in an ice-water slurry

Stirred for 20 min after all the salt had been dissolved

Kept undisturbed overnight at 4 °C

Sample centrifuged (3000 x g, 20 min, 4 °C), pellet dissolved in PBS

Lectin titre and protein content of supernatant and dissolved pellet was quantified
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 – Dialysis
Obtained after 40% saturation of ammonium sulphate was loaded onto dialysis tubing
(10 KDa)

Dialysed against 0.1M PBS, pH 7.2 at 4 °C for 24 h with three buffer changes every 8 h

Lectin titre and protein content of the dialysed sample was quantified

 Characterization of the partially purified lectin:

 Optimum pH – PBS set at pH 4.5-9.0.

 pH stability – sample incubated in PBS (pH 4.5-9.0), lectin activity

assayed at 0 min and subsequently at 15 min intervals upto 2 h.

 Optimum temperature – 25-70 °C.

 Thermal stability – 500 µl lectin sample incubated over a range of 25-

70 °C, lectin activity assayed at 0 min and subsequently at 15 min
intervals upto 2 h.
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 Protein Estimation:

Reagents

Solution A: 2.0% Na2CO3 prepared in 0.1N NaOH.

Solution B: 0.5% CuSO4 prepared in 1.0% sodium potassium tartarate.
Solution C: 50 ml of solution A and 1 ml solution B (prepared fresh).
Solution D: Folin-ciocalteau’s reagent: distilled water (1:1, prepared fresh)

Procedure

To 1 ml app. Diluted sample , 5 ml of solution C was added

Mixed, incubated at RT for 10 min

0.5 ml solution D was added, incubated for 25 min at RT

Absorbance noted at 660 nm

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 0.9

0.8

0.7

0.6

Absorbance (660 nm)

0.5

0.4

0.3

0.2

0.1

0
0 100 200 300 400 500 600
Bovine serum albumin (ppm)

Fig. 3.1: Standard curve for protein estimation

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RESULTS

Table 4.1: Biological action spectrum of lectin produced by A. niger.

Human
Goat Pig Rabbit Mice Rat
A B AB O

+ + + + - + + - -

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Plate 4.1: Biological action spectrum of lectin produced by A. niger.

A, B, AB, O: Human

Rb : Rabbit

Ra : Rat

Mi : Mice

Go : Goat

Pg : Pig

C: Control

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Table 4.2: Agglutination of lectin produced by A. niger with cells.

Cells Agglutination

Escherichia coli -

Kluyveromyces marxianus -

Splenocytes -

Lymphocytes +

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 300

250

200

Titre
150

100

50

0
Untreated Protease treated Neuraminidase
treated

Fig. 4.1: Effect of enzymatic treatment of erythrocytes on their agglutination by A. niger lectin.

22
 140

120

100

80

Titre 60

40

20

0
Solidified Broth

Fig. 4.2: Lectin activity as a function of mode of cultivation.

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 Specific sugars Non-Specific sugars
D-ribose (well 1) D-raffinose (well 3)
L-rhamnose (well 2) D-xylose (well 4)
L-fucose (well 5) D-mannose (well 6)
D-arabinose (well 7) D-galactose (well 8)
D-sucrose (well 9) D-glucose (well 10)
D-fructose (well 12) D-maltose (well 11)
D-mannitol (well 13) D-lactose (well 14)
Bovine submaxillary mucin (well 16) Inulin (well 15)
Asialofetuin (well 17) Pullulan (well 19)
Chondroitin-6-sulphate (well 18) Inositol (well
20)
D-galactosamine hydrochloride (well 25) Meso-inositol (well
21)
N-acetyl-D-galactosamine (well 29) L-arabinose (well
22)
2-deoxy-D-glucose (well 30) D-trehalose dihydrate (well 23)

Melibiose (well 33) D-glucosamine hydrochloride (well 24)

Plate 4.2: Sugar inhibition profile of
γ-globulin (well 36) D-glucuronic acid (well 26)
A. niger lectin.
Porcin stomach mucin (well 37) D-galacturonic acid (well 27)
N-acetyl-D-glucosamine (well 28)

2-deoxy-D-ribose (well 31)

Thiodigalactoside (well 32)
Starch (well
34)
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Table 4.4: Minimum inhibitory concentration (MIC) of specific sugars.

Sugar MIC

D-ribose >12.5 mM
L-rhamnose >50 mM
L-fucose >25 mM
D-arabinose >25 mM
D-fructose >12.5 mM
D-mannitol >50mM
D-sucrose >12.5 mM
Melibiose >50 mM
D-galactosamine hydrochloride >31.75 µg/ml
N-acetyl-D-galactosamine >6.25 mM
2-deoxy-D-glucose >0.19 mM
Chondroitin-6-sulphate >0.12 µg/ml
Bovine submaxillary mucin >62.5 µg/ml
Porcine stomach mucin >125 µg/ml
Asialofetuin >125 µg/ml
γ – globulin >125 µg/ml

25
 1200

1000

800

Titre
600

400

200

0
3 4 5 6 7 8 9 10 11
Growth (days)

Fig. 4.3: Lectin activity as a function of growth.

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 Table 4.5: Optimization of ammonium sulphate precipitation of A.niger lectin.

Percent Volume Protein Total Titre Total titre Specific

saturation of (ml) (mg/ml) protein activity
ammonium (mg) (Titre/mg)
sulphate

0 10 16.079 160.789 256 2560 15.921

30 2 26.947 51.894 64 128 2.375
40 2 31.684 63.368 1024 2048 32.319
50 2 39.105 78.210 1024 2048 26.186
60 2 42.842 85.684 1024 2048 23.902
70 2 44.000 88.000 1024 2048 23.272
80 2 47.210 94.421 1024 2048 21.690
90 2 45.684 91.368 1024 2048 22.415

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 Table 4.6: Summary of partial purification of lectin produced by A. niger.

Sample Volume Titre Total Protein Total Specific Fold Yield

(ml) titre (mg/ml) protein activity Purificatio (%)
(mg) (Titre/mg) n

Crude 100 256 25600 15.684 1568.4 16.322 1 100

Precipitate 20 1024 20480 28.421 568.420 36.029 2.20 80
Dialysate 25 512 12800 18.683 467.075 27.404 0.76 50

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 600

500

400

Titre
300

200

100

0
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
pH

Fig. 4.4: Optimum pH for lectin activity of A. niger.

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 70

60

50

40
Titre

30

20

10

0
0 15 30 45 60 75 90 105 120
Time (min)

4.5 5 5.5 6 6.5 7 7.5 8 8.5 9

Fig. 4.5: pH stability of partially purified lectin from A. niger.

30
 600

500

400
Titre

300

200

100

0
25 30 35 40 45 50 55 60 65 70

Temperature ( o C)

Fig. 4.6: Optimum temperature for lectin activity of A. niger.

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 600

500

400

Titre
300

200

100

0
0 15 30 45 60 75 90 105 120
Time (min)

30 °C 35 °C 40 °C 45 °C 50 °C 55 °C
60 °C 65 °C 70 °C

Fig. 4.7: Thermal stability of lectin produced by A. niger.

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CONCLUSIONS

 Agglutination observed with all human blood types, rabbit,

pig erythrocytes and lymphocytes.

 Highest lectin activity observed in 9-10 days old cultures.

 Lectin activity was 4 fold higher in broth cultures as

compared to solidified medium.

 The lectin showed very high affinity to chondroitin-6-sulphate

(>0.12 µg/ml) and bovine submaxillary mucin (>62.50 µg/ml).

Contd .
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 Optimum concentration of ammonium sulphate - 40%
saturation.

 Specific activity obtained in precipitates - 32.319 titre/mg with

2.20 fold purification.

 Specific activity of dialysate exhibited - 27.404 titre/mg.

 Optimum pH for the lectin activity - 7.5-8.0 .

 Optimum temperature for lectin activity - 35-40 oC.

 The lectin was found to be stable within the pH range of 7.0-

8.0 even after 2 h.

 Lectin retained activity even after 2 h upto 40 °C.

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