impact of cultural and nutritional conditions on l-asparaginase … · 2015. 4. 12. · et al.,...

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Int. J. Pharm. Med. & Bio. Sc. 2014 Nibha Gupta and K Krishna Raju Patro, 2014

IMPACT OF CULTURAL AND NUTRITIONAL

CONDITIONS ON L-ASPARAGINASE

PRODUCTION BY PENICILLIUM CITRINUM THOM.

K Krishna Raju Patro1 and Nibha Gupta1*

Research Paper

In the present study production of L-asparaginase enzyme by Penicillium citrinum Thom. wasdetermined in different culture and nutritional conditions. The fungal culture preferred fructoseand ammonium nitrate for better enzyme production. Finally, the L- asparaginase extractedfrom cell biomass was purified through gel filtration and ion exchanged chromatography andcharacterized for its purity, molecular wt., thermal tolerance, pH requirement. Gel filtration andion exchange chromatography produced pure enzyme which was later confirmed through PAGEexhibited 29.0 kDa. The partially pure enzyme preferred 6.0-7.2 pH, enzyme activity lowereddown in higher pH. It was thermally stable under the treatment of 45-50 C. All experiments hadbeen done under batch culture. However, the medium composition acquired finally can be usefulfor the continuous fermentation for large scale production of L - asparaginase.

Keywords: L-asparaginase, Penicillium, Gel fitration

*Corresponding Author: Nibha Gupta � [email protected]

INTRODUCTION

The L-asparaginase enzyme hydrolyse the L-

asparagine into L-asparatic acid and ammonia

and found to be promising candidate for the

treatment of acute lymphocytic leukemia .

Microbes are better source of this enzyme,

because they can be cultured easily and

extraction and purification of L- asparaginase from

them is also convenient ( Savitri et al., 2003)..

Many bacteria and fungi are studied for the

production of this enzyme (El Bessoumi et al.,

2004; Sahu et al., 2007; Gupta et al., 2009; Shah

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Int. J. Pharm. Med. & Bio. Sc. 2014

1 Division of Plant Pathology and Microbiology, Regional Plant Resource Centre, Bhubaneswar , Odisha 751-015.

et al., 2010). Many fungal strains have been

reported well for the potential of L- asparaginase

production (Theantana et al., 2007; Gupta et al.,

2009; Lapmak et al., 2010, Venil et al., 2009 ).

Some fungi as Aspergillums tamari, Aspergillums

terreus have proved to be beneficial sources of

this enzyme (Nakahama et al., 1973; Jayaramu

et al., 2010; Siddalingeshwara and Lingapp,

2010). In order to use this enzyme as health care

agent, large scale production is needed that

depend upon the source of enzyme, better culture

and biochemical environment and extraction

protocols Baskar et al., 2009 ). In view, a

115


systematic study has been planned to upscale

the production of L-asparaginase in Penicilluim

citrinum by supplementing the basal media with

different nutritional factors.

MATERIALS AND METHODS

Penicillium citrinum Thom. used was obtainedfrom the culture collections of MicrobiologyLaboratory of Regional Plant Resources Centre,Bhubaneswar, Odisha. The fungal culture wasprepared in glucose – asparagine broth (Glucose– 1%, L asparagine-0.5%, KH

2PO

4 -0.1% and

MgSO4-0.05%, pH 4.5) by growing at 30C for 10days in static condition. Similarly, the fungus wasgrown with different carbon sources and nitrogensources by keeping basic constituents constant.Effect of salt and ionic compounds was studiedby adding them individually in medium containingselected carbon and nitrogen sources along withKH2PO4 and MgSO4 (0.1%). The pH optima andsuitable incubation period for the enhancedenzyme production was determined by cultivatingthe fungus at different pH level ranged between4-9 and incubated 4 days interval up to 16. Thecells were harvested, homogenized with 0.5 MTris-HCl buffer (pH-8.5) and centrifuged at 3000rpm for 20 min. The supernatant was used forammonium sulphate precipitation with 80%saturation. The enzyme was assayed bymeasuring the amount of ammonia liberated fromL- asparagines (Baskar and Renganathan, 2009).The enzyme activity was expressed in terms ofenzyme units (IU/mL) or international units.Sephadex G-100-120 (Sigma) was used for thegel filtration and ion exchange chromatography.Gel electrophoresis was performed (at 40 V for 1h and then 55 V for 3 h) to estimate the molecularweight. Finally, purified enzyme wascharacterized for the substrate specificity, pHoptima, and thermal tolerance.

RESULTS AND DISCUSSION

The fungal culture preferred shake condition

(@50rpm) and produced more amount of enzyme

when grown in basal glucose asparagines

medium of 4.5 pH at 30 ºC for 8 days. Among

three carbon sources used in different

concentration under this experiment, fructose

exhibited enhancement in enzyme activity at 20-

25 g/L concentration. However, aesculin and

lactose showed good effect on L asparaginase

production. Hence, 20 g/L fructose in basal

medium were selected for further experimentation

(Figure 1).

Different amino acids, viz., asparagine,

tryptophan, glutamine, cysteine, proline,

threonine, methionine, phenylalanine, alanine,

tyrosine, arginine, aspartic acid were added

separately into the basal medium along with the

selected carbon source (Fructose 1%) to observe

their effect on L asparagines production. Addition

of trytophan exhibited maximum enzyme activity

(Figure 2). Other compounds tested as nitrogen

source did not show better effect on enzyme

activity (Figure 3) .The basal medium having 2%

fructose and added separately with different

Figure 1: Selection Of Carbon SourceFor Better L Asparaginase Production

(1% Carbon Source)

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concentration of (0.5, 0.6, 0.7, 0.8 and 0.9 %) L

asparagines and tryptophan did not show much

effects on enhancement in enzyme activity.

Hence, 0.5% concentration of L asapragine and

tryptophan were used in further experimentations.

The basal medium having 2% fructose added

separately with ammonium chloride, ammonium

nitrate and glycerol @ 0.5% concentration

exhibited effective response of ammonium nitrate

in enhancement of enzyme activity (Figure 4).

The basal medium added with Fructose (2%),

tryptophan (0.5%) , Ammonium nitrate (0.5%)

used in flasks of different sizes (100, 150, 250,

and 500 mL) did not exhibited much difference in

enzyme activity. However, container of 250 mL

showed better enzyme production. Similarly,

lower speed (50 rpm ) of shaking found to be good

as compared to 60 rpm (Figure 5).The profile of

the ammonium sulfate fraction purification on

Sephadex (G100-120 gel filtration column

chromatography exhibited that the fraction

contained different protein molecules, fraction 10

to 20 showed highest enzyme activity .Similarly,

fractions of sephadex G 100 gel filtration on the

DEAE column showed the most possible

presence of enzyme in fraction no 2- 4. Gradually

the protein as well enzyme content of further

fractions was decreasing.

Figure 2: Effect Of Different Amino Acidson L asparaginase

Figure 3: Effect of Other Compounds(Fructose 1%+4 days+shaking+basal medium)

Figure 4: Effect of Additional Compoundon L asaparaginase Activity

Figure 5: Effect Of Shaking Speed AndCulture Flask Volume (2% Fructose+0.5%

Tryptophan + Amm Nitrate 0.5%+4 Days + Basal Medium)

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The partial purification of the L asparaginase

crude extract that was affected by the ammonium

sulfate *80%) precipitation showed that most of

the enzyme activity was preserved in the

precipitate. The total protein decreased from 22.4

mg to 10.2 mg in the ammonium sulfate

precipitation steps. The specific activity increased

to 463.647 IU/mg and 1176.458 IU/mg after

ammonium sulfate and sephadex gel filtration

steps, respectively. Partially purified enzymes

obtained from sephadex G-100 and DEAE

column chromatography was characterized for

the effect of pH, temperature and substrate

specificity. It prefers the alkaline pH ranged 6-7.2.

The enzyme activity was lowered at high pH (Table

1). Sephadex purified enzyme was active at 45-

50 C where as DEAE separated enzyme was

gradually decreasing its activity at higher

temperature (Table 2). Partially purified enzyme

(sephadex-G-100-120) exhibited preference to

asparagines, aspartic acid, arginine and histidine

and glutamic acid for substrate requirement.

Whereas DEAE purified protein exhibited good

activity with asparagines, arginine, histidine, and

glutamic acid (Table 3). Standardization of

electrophoretic separation indicated the purified

protein and enzyme preparations are matched

with 29 kDa.

Penicillium citrinum Thom. has given

preference shake culture process for the better

enzyme production. The fungus was stimulating

the enzyme production in batch culture also

supports the studies made by Liu and Zajic (1973)

who stated that batch culture is better than the

continuous fermentation process in Erwinia

aroideae. The fungus gave preference to fructose

for the enhanced enzyme production where as

glucose showed less enzyme production. This

result was also corroborated with the studies of

Tosa and Sano (1971) done on effect of substrate

Table 1: Purification Profile of L asparaginase from

Collected Total Total Specific Purification

volume activity protein activity (Fold) Yield (%)

(ml (IU) (mg) (IU/mg)

Crude extract 280 757.4 22.4 33.812 0 100

Ammoniumsulphate ppt 60 4729.2 10.2 463.647 13.712 624.399

Gel filtration Sephadex G 100 80 2258.8 1.92 1176.458 34.793 298.125

DEAE Cellulose 10 278.81 0.31 899.387 26.599 36.811

Table 2: Effect of pH on L asparaginaseActivity (Iu/ml)

pH Sephadex gel filtration DEAE cellulose

4 30 17.352

6 25.883 26.529

8 21.47 27.647

10 17.647 23.529

7.2 27.176 27.294

Table 3: Effect of Temperatureon L asparaginase Activity (Iu/ml)

Temperature (oC) Sephadex DEAE

30 27.352 33.529

37 27.352 33.529

45 30.588 29.117

50 30.882 27.264

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for the higher production of L-asparaginase by

some bacteria (Table 4). The addition of glucose

to the medium resulted in depressed production

of enzyme. It also explain the synthesis of L –

asparaginase in Penicillium citrinum Thom. Strain

H was almost suppressed in presence of

glucose. It may be due to the catbolite repression

and catabolic inhibition caused by glucose, as

regulatory biomolecule (Warrykar and

Khobragade, 2009). Preference of Penicillium

citrinum Thom. Strain H for the use of fructose

as best carbon source for good enzyme

production may be species specific. The use of

a relatively poor carbon source like mannitol

caused a lower L – asparaginase level (Geckil

et al., 2004).

Growth and metabolism may be regulated by

different nutritional factors and species dependent

(Barnes et al., 1977). The fungal strain Penicillium

citrinum Thom. Strain H was supplied various

nutritional and environmental factors for the

improvement in enzyme production in order to

obtain standardized cultural parameters for the

large scale fermentation. In present study

organisms preferred 4.5 acidic p\h to give best

enzyme production and evaluation of enzyme

production at different incubation period showed

to early stages. Methew et al. (1994) reported that

nitrogen is also limiting factor. It is the reason ,

some time addition of amino acid suppress the L

–asparaginae activity . Penicillium citrinum Thom.

Strain H was found to be induced by the presence

of tryptophan in the culture medium. The

interaction of carbon sources and ammonium

salts is well exhibited in the present study. In certain

ratio, the L-asparagine and ammonium nitrate

encourages high production of enzyme in this

fungus. However, glycerol did not affect much in

this regard. The low molecular wt. of partially

purified enzyme from Penicillium citrinum Thom.

did not showed the very high potential of this

organism to be exploited. However, its tolerance

of low pH and thermal stability should be under

consideration for the further drug development

program.

ACKNOWLEDGMENT

The support received from Ministry of Earth

Sciences, Govt. of India under the research

project no. No. MoES/11/MRDF/1/30/P/08 is

gratefully acknowledged.

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impact of cultural and nutritional conditions on l-asparaginase … · 2015. 4. 12. · et al.,...

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