6 purification and characterization of l-...

93 Purification and characterization of L- Asparaginase

6 Purification and characterization of L-

Asparaginase

6.1 Introduction

Purification of a protein is an important step for characterization of

its physical and biological properties. Moreover, for effective

therapeutic use of a protein, it must be free of any contaminants

and impurities.

Success of a downstream process mainly depends on the cost

effectiveness, less sequential operations and overall yield of the

product. Cost and yield of the product is mainly influenced by the

number of sequential steps involved in purification. Downstream

processing accounts for around 80% of overall production cost,

which clearly indicates a need for process optimization. Above all,

the purification process must be simple, easy and adaptable,

particularly in large scale (125).

Cytotoxicity studies are the preliminary step in anticancer drug

screening. Many methods are available for screening the anti-

cancer potential of agents viz., tryphan blue dye exclusion assay,

microculture tetrazolium assay (MTT) and sulpho rhodamine assay

( SRB). The antileukemic effect of L-asparaginase is postulated to

result from the rapid and complete depletion of the circulating pool

of asparagine. Cytotoxicity is a result of depletion of non- essential

amino acid, asparagine. The leukemic cell has repressed asparagine

synthetase activity, whereby they depend on the circulating

asparagine.

The current study describes the methods adopted and results of

purification, characterization and in vitro anti-leukemic activity of

L-asparaginase produced by isolate SI099 (Aspergillus terreus).

94 Exploration of soil and marine sources for microbes producing asparaginase

6.2 Materials and Methods

6.2.1 Production of L-asparaginase by submerged fermentation

Production of L-asparaginase was carried out in Erlenmeyer flask

containing optimized medium (Table 6-1) for 5 days at 165 rpm

and 25ºC with 7.5 % inoculum. The cell free supernatant was

collected by centrifuging at 1500 rpm for 15 min and was used for

estimating the extracellular enzyme activity. Enzyme assay was

carried out as described in section 4.2.3. The protein content was

estimated by Lowry method(126). Specific activity of the enzyme

was expressed as IUmg-1

protein. (Materials used are listed in

appendix)

Table 6-1: Optimized medium used for production of L-asparaginase

S.No Ingredient gL-1

1 Sucrose 16.1

2 Yeast extract 18.0

3 Ammonium chloride 10

4 Magnesium sulphate 0.5

5 L-Asparagine 31.9

pH adjusted to 8.0

6.2.2 Purification of L-asparaginase

The sequential steps followed in purification included ammonium

sulfate precipitation, ion exchange and gel filtration

chromatography. The molecular weight of the L-asparaginase was

determined by Sodium Dodecyl Sulphate Poly Acrylamide Gel

Electrophoresis (SDS-PAGE).


6.2.2.1 Ammonium sulfate precipitation

Ammonium sulfate removes water from the surrounding of the

protein revealing hydrophobic patches, which come together and

causes the protein to precipitate. The more a protein is hydrophilic,

the more will be the ammonium sulfate needed. The fractionation

range of ammonium sulfate needed to precipitate out the target

protein was determined by performing analytical ammonium

sulfate cut (127, 128).

Ammonium sulfate was added to the supernatant in different

concentrations ranging from 10 to 90%w/v saturation, with

constant stirring in ice bath. The precipitate was removed by

centrifuging at 10000 rpm for 10 min in cooling centrifuge

maintained at 4°C. The supernatant was used for estimation of

enzyme activity and protein content. The fractionation range of

ammonium sulfate needed was determined. The fractionation range

of ammonium sulfate was found to be 40-70% w/v.

The precipitation of the target protein was done using the

fractionation range. Initially the cell free supernatant was brought

to 40% saturation with ammonium sulfate and kept at 4-8°C

overnight. After overnight equilibration, the precipitate was

removed by centrifuging at 10000 rpm for10 min in cooling

centrifuge at 4°C. The supernatant was further brought to 70%

saturation with ammonium sulfate and left overnight at 4-8°C. The

precipitate was collected by centrifuging at 10000 rpm and 4 °C for

15 min.

The precipitate was re-suspended in 10 mL cold 50mM Tris-HCl,

pH 8.6 and desalted using sephadex G-25 column with the same

buffer. An aliquot from this was used to determine the enzyme

activity and protein content. The desalted protein solution was

collected, stored at 4-8°C and used in further steps of purification.


6.2.2.2 Ion exchange chromatography (IEC)

The sample obtained after desalting was diluted to 50mL of 50mM

Tris-HCl buffer and used in IEC. DEAE-sepharose anion

exchange column was equilibrated with 50mM Tris-HCl buffer

(pH 8.6). To the equilibrated column, the sample was applied and

the column was washed with two bed volumes of the same buffer

to remove any unbound protein.

The sample was eluted as 6mL/fraction using NaCl gradient (0.1-

0.5M) at flow rate of 30mLh-1

. The protein content and enzyme

activity were determined. The fractions showing peak L-

asparaginase activity were pooled together and concentrated by

dialysis against poly (ethylene glycol) 20000, followed by dialysis

against 50mM Tris –HCl buffer at 4°C (129, 130).

6.2.2.3 Gel filtration chromatography (GFC)

The sample obtained after dialysis was chromatographed on a

column of Sephacryl S 200, which was pre-equilibrated with

0.05M Tris-HCL buffer of pH 8.6. The sample was eluted with the

same buffer at 24mLh-1

flow rate as 4mL fractions. The enzyme

activity and protein content of the fractions were determined.

Fractions with peak enzyme activity were pooled together and

concentrated by dialysis against poly (ethylene glycol) 20000,

followed by dialysis against 50mM Tris –HCl buffer and stored at

4-8°C (131).

6.2.2.4 Determination of molecular weight of enzyme by SDS-PAGE

The sample after purification steps was electrophoresed by SDS-

PAGE (132), with 5% stacking gel and 10% resolving gel.

Standard markers included phosphorylase (97.4kDa), bovine serum

albumin (66.2kDa), ovalbumin (43kDa), carbonic anhydrase

(31kDa), trypsin inhibitor (20.1kDa) and lysozyme (14.3kDa).


6.2.3 Partial characterization of the enzyme

6.2.3.1 Effect of metal ions for the enzyme activity

The effect of different metal ions on the enzyme was studied by

pre-incubating enzyme with 10mM zinc sulphate, calcium chloride,

sodium chloride, potassium chloride, ferrous sulphate and

magnesium sulphate for 1h at 37°C. The relative enzyme activity

was estimated.

6.2.3.2 Effect of inhibitors and chelators on enzyme activity

The effect of different inhibitors viz., phenyl methyl sulphonyl

fluoride (PMSF, 5mM), dithiothritol (DTT, 5mM), β-

mercaptoethanol (MCE, 5mM) and chelator viz., 5mM ethylene

diamine tetra acetic acid (EDTA) was determined. The enzyme

was pre-incubated with respective inhibitor or chelator at 37°C for

1h and then the enzyme assay was performed under standard

conditions.

6.2.3.3 Effect of pH on enzyme activity and stability

The effect of pH on enzyme activity was studied by estimating the

activity at pH ranging from 4.0 to 12.0 with different buffers viz.,

phosphate buffer mixed (pH4.0), phosphate buffer ((pH 5.0-7.0),

Tris-HCl (pH 8.0) and glycine-NaOH (pH 9.0-12.0).

The enzyme was incubated in contact with different relevant

buffers (pH 7.0-12.0) at 37°C for 48h to determine its stability in

different pH. The relative activities were measured.

6.2.3.4 Effect of temperature on enzyme activity and stability

The effect of temperature on enzyme activity was determined by

incubating the reaction mixture at different temperatures ranging

from 20 to 90°C.


The stability of enzyme in different temperature was studied by

incubating the enzyme at different temperatures for 1 h. The

residual enzyme activities were determined.

6.2.3.5 Determination of substrate specificity and kinetic parameters

The enzyme activity was determined with L-asparagine and L-

glutamine as substrate at a final concentration of 10 mM. High

performance thin layer chromatography of the digest was

performed.

The Michaelis constant (Km) and maximal velocity (Vmax) of the

purified enzyme were determined using L-asparagine as substrate

in the range of 50–1600 µM with the help of Lineweaver-Burk

plot. (133-135)

6.2.4 In vitro anti-leukemic activity of the purified L-asparaginase

by MTT assay (136)

The human ALL cell line MOLT-4 was cultivated as suspension

culture in RPMI 1640 medium supplemented with 10%(v/v) fetal

bovine serum (FBS) at 37 °C in a 5% CO2 incubator.

The effect of L-asparaginase on the growth rate of MOLT-4 cells

was determined by MTT. Briefly, exponentially growing cells

were collected by centrifugation for 10 min at 2000 rpm, washed

twice in PBS, and re-suspended at a density of approximately

4x105 cells/mL in fresh medium. The cells were seeded in 96-well

plates at a density of 4000 cells/well in RPMI 1640 medium

supplemented with 10%(v/v) fetal bovine serum (FBS) and

incubated at 37 °C for 24 h in a 5% CO2 incubator. After 24 h,

the plates were centrifuged at 2000 rpm for 10 mins and the

supernatant was carefully replaced with 100 µL of medium

containing purified L-asparaginase at different concentrations

(0.01, 0.1, 1, 10 IU).


After incubation for 48 at 37 °C and 5% CO2, the plates were

centrifuged at 2000 rpm for 10 mins and inverted on tissue paper to

remove the media. The cells were washed once with 100 µL PBS

and the supernatant were removed after centrifugation at 2000 rpm

for 10 mins.

To each well 100 µL MTT was added (2mg/mL) and the cells were

incubated for an additional 2 h. Later, the formazan crystals formed

were dissolved in 100 µL of isopropanol and incubated at 37 C for

30 mins. The optical density was measured at 540nm using Biotek

ELx plate reader. The experiments were performed as duplicates

and the percentage growth inhibition was calculated.

6.3 Results

6.3.1 Purification of L-asparaginase

Activity guided analytical ammonium sulphate cut method was

used to determine the fractionation range for precipitation of the

target protein. The fractionation range was found to be 40-70%w/v

saturation of ammonium sulfate.

With preparative ammonium sulphate precipitation, the yield was

58.03 and purification fold was 11.02. The desalting was

performed using sephadex G25 and the elute was diluted to 50mL

with 50mM Tris-HCl buffer (pH 8.6) and further purified by IEC.

The chromatogram of IEC was given in Chart 6-1. Five different

protein peaks were observed and the target protein was eluted with

0.2M NaCl (Fraction no.12-16). The results of purification are

given in Table 6-2. The purification fold and yield after IEC was

found to be 23.08 and 34.95, respectively.

Elute from IEC was subjected to dialysis and concentrated. It was

further purified by GFC and the chromatogram was given in Chart

6-2. The target protein was eluted in three fractions (10-12). The


yield and purification fold of the pooled fractions was found to be

24.58 and 86.75, respectively.

SDS-PAGE of the purified sample was performed and the results

were given in Figure 6-1. The molecular weight of the denatured

enzyme was calculated from the Rf values and was found to be

35.26 kDa (approx. 35kDa).

6.3.2 Partial characterization of L-asparaginase

The effect of pH, temperature, metal ions, inhibitors and chelators

on the enzyme activity was studied. The results are given as Charts

6-3 to 6-6. Among the metal ions tested, enzyme activity was

greatly affected by ferrous ions. Sodium, potassium and calcium

ions enhanced the enzyme activity. The enzyme was found to be

inhibited by PMSF (serine protease inhibitor) and DTT (cysteine

protease inhibitor).

The enzyme was found to be stable at wide pH of 7-11.0. There

was 20% decrease in activity when stored at pH 12.0 for 48h. The

optimum pH for enzyme activity was found to be pH 8.0.

The enzyme was stable when stored at varied temperatures ranging

from 20 to 50°C. Storage temperature above 50°C affected the

enzyme activity. The optimum temperature for enzyme activity

was found to be 40°C.

The relative activity of the enzyme using L-glutamine was found to

be 7%. The result of HPTLC was given in Figure 6-2 and the Rf

values are given in Table 6-3. The Km and Vmax of the enzyme was

found to be 277.78 µM substrate and 40.39 µM/min (See Charts 6-

7 & 6-8).


6.3.3 In vitro anti-leukemic studies

The percentage inhibition of cell proliferation by L-asparaginase at

different concentrations was calculated. The results were expressed

as mean values in Table 6-4. The concentration of L-asparaginase

causing inhibition of 50% of viable cells (IC50) was calculated.

The results were compared with marketed formulation of L-

asparaginase.


Chart 6-1 Ion Exchange Chromatography: Elution Profile

Chart 6-2 Gel Filtration Chromatography: Elution Profile

0

100

200

300

400

500

0

1

2

3

4

5

0 5 10 15 20 25 30 35 40 45 50

En

zym

e a

cti

vit

y (

IU/m

L)

Prote

in (

mg/m

L)

Fraction No (5mL each)

Protein

Enzyme

0

100

200

300

400

500

0

1

2

3

4

5

0 5 10 15 20 25

En

zy

me a

cti

vit

y (

IU/m

L)

Prote

in (

mg

/mL

)

Fraction No (4mL each)

Protein

Enzyme


Chart 6-3 Effect of metal ions on the enzyme activity

Chart 6-4 Effect of inhibitors and chelators on enzyme activity

0

20

40

60

80

100

120

Rela

tive E

nzym

e A

cti

vit

y (

%)

Metal Ions (10mM)

Calcium Magnesium Zinc Ferrous Sodium Potassium

0

10

20

30

40

50

60

70

80

90

100

PMSF MCE DTT EDTA

Rela

tive a

cti

vit

y (

%)


Chart 6-5 Effect of pH on stability and activity of enzyme

Chart 6-6 Effect of temperature on stability and activity of enzyme

0

20

40

60

80

100

120

4 5 6 7 8 9 10 11 12

Rela

tive a

cti

vit

y (

%)

pH

Activity Stability

0

20

40

60

80

100

120

20 30 40 50 60 70 80 90

Rela

tive A

cti

vit

y (

%)

Temperature (°C)

Activity

Stability


Chart 6-7 Plot of reaction velocity (v0) vs. Substrate concentration [S]

Chart 6-8 Lineweaver-Burk plot

0

1

2

3

4

0 200 400 600 800 1000 1200 1400 1600 1800

v0 (

µM

/min

)

[S] (µM)

y = 68.842x + 0.2476

R² = 0.9885

-1.5

-1

-0.5

0

0.5

1

1.5

2

-0.03 -0.02 -0.01 0 0.01 0.02 0.03

1/v

0

1/S


A-Elute from IEC; B- Elute from GFC, M-Marker

Figure 6-1 Silver stained SDS-PAGE: Molecular weights of the bands

Figure 6-2: HPTLC of the digests


Table 6-2: Results of Purification of L-asparaginase

S.No Purification Step

Total

Enzyme

activity(IU)

Total Protein

(mg)

Specific Activity

(IUmg-1

protein)

Purification

Fold Yield

1 Crude 15450 4870 3.1724846 0 100

2 (NH4)2SO4 8965 256.5 34.95126706 11.01700133 58.02589

3 DEAE-Sepharose 5400 73.75 73.22033898 23.07980912 34.95146

4 Sephacryl 200HR 3798 13.8 275.2173913 86.75137189 24.58252

Table 6-3: Rf values observed in HPTLC

Track Spot Peak Rf

1 L-Asparagine 2 0.35

3 L- Glutamine 1 0.44

4 L-aspartic acid 2 0.37

5 L-glutamic acid 1 0.45

6 Digest- L-Asparagine 1 0.38

7 Digest- L-Asparagine 1 0.38

8 Digest- L-Glutamine 1 0.43

9 Digest- L-Glutamine 2 0.43


Table 6-4: Percentage of Growth inhibition in MTT assay

S.No

Enzyme

concentration

(IUmL-1

)

Growth Inhibition (%)

Purified L-

asparaginase

from SI099

Marketed L-

asparaginase

preparation

1 20 9 12

2 40 12 16

3 60 22 27

4 80 37 45

5 100 64 79

IC50 91.41 IUmL-1

77.42 IUmL-1

6.4 Discussion

Purification of L-asparaginase was carried out using appropriate

chromatographical techniques. The specific activity before

purification was 3.17 IUmg-1

protein. Upon precipitation with

ammonium sulphate the specific activity was found to be increased

by 11 fold and the total protein content was found to be decreased

by 19 fold. This indicates that the fractionation range of

ammonium sulfate used for precipitation was effective enough in

removing proteins which are contaminants, with subsequent loss of

total activity (approximately 42%).

With IEC, only 1.5% of the total protein from crude extract was

eluted (66 fold decrease), but the specific activity was found to be

increased by 23 fold. The specific activity after IEC was 73.22

IUmg-1

protein. Five different protein peaks observed in the

elution profile indicates an efficient removal of contaminants.

GFC was performed as final step of purification. The specific

activity after GFC was found to be increased by 86.75 fold, with

total protein content decreasing by 352 fold. Three bands were

observed in SDS-PAGE after IEC. And after GFC, only one band

was observed. From the bands observed, it can be claimed that the

enzyme was purified to near homogeneity.


The enzyme activity was severely affected by PMSF, a serine

protease inhibitor and DTT, a cysteine protease inhibitor. The

stability of enzyme at varied pH and temperature reduces the cost

involved in storage and transportation. The low Km value

indicates that the enzyme has high specificity for the substrate L-

asparagine. The results of chromatography indicate no or little

effect on L-glutamine. Overall, the enzyme has high L-

asparaginase activity and no or little L-glutaminase activity.

The IC50 value of L-asparaginase from isolate SI099 is

comparable with the marketed L-asparaginase preparation. The

IC50 values were quite high when compared to earlier results on

HL60 cell lines with different L-asparaginase.

6.5 Conclusion

The sequential purification of L-asparaginase from isolate SI099

(Aspergillus terreus) resulted in 86.75 fold pure enzyme. The

molecular weight of the purified denatured enzyme was found to

be 35.26 kDa. The enzyme was considerably stable at pH range 7-

10.0 and temperature range 20-50°C. The peak activity was at pH

8.0 and 40°C. The enzyme was found to be substrate specific to L-

asparagine with 6% relative activity with L-glutamine as substrate.

The purified L-asparaginase from isolate SI099 has an IC50 value

of 91.41 IUmL-1

6 purification and characterization of l-...

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