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RESEARCH ARTICLE

ARTICALTICLE

International Journal of Pharma and Bio Sciences

A KINETIC STUDY ON CELLULASE ENZYMES FROM ASPERGILLUS NIGER

¹SAXENA ARTI , ²SAXENA ANURAG AND ¹ YADAV SARIKA

¹ Deptt. of Chemistry , A.N.D.N.N.M.College, Kanpur.

² Deptt. of Physics, D.A-V. College,Kanpur.

*Corresponding author

BIO CHEMISTRY

ABSTRACT

A physico-chemical study was made on cellulase enzymes from a highly cellulolytic fungus Aspergillus niger. Various kinetic parameters were estimated by making use of potent fungicides, viz. m-dinitrobenzene and pentachlorophenol. The enzyme showed maximum activity at pH 5.0 and temp. 55o C. The half-life was found to be 42 minutes at optimum pH. The Michaelis Constant, Km, was observed as 13.3mM and Vmax. as 6.65µM/sec. The inhibition constant, Ki, for MDB was observed to be 19.2mM and for PCP as 22.6mM, thereby indicating that MDB is a more potent inhibitor than PCP.

SAXENA ARTI Deptt. of Chemistry , A.N.D.N.N.M.College, Kanpur.

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KEYWORDS

Biodegradation, fungicides, inhibitive

INTRODUCTION Cellulase enzymes from various micro-organisms, mainly fungi, act on cellulosic materials and biodegrade them. Cellulase is a synergistic enzyme that is used to break up cellulose into glucose or other oligosaccharide compounds (5). Incorporation of suitable toxic substances in the cellulosic materials may prevent them from microbial attack. The mechanism of fungicidal action on cellulolytic micro-organism was studied to facilitate the problem of selecting suitable fungicides for the protection of stores, such as, textiles, timber, leather etc. against microbial attack. For this purpose a highly cellulolytic fungus Aspergillus niger was selected and kinetic studies were made. The present work deals with the

physico-chemical studies on cellulase enzymes elaborated by the fungus.

MATERIALS AND METHODS A. niger was grown in Czapek’s medium. The culture was incubated at 30 + 2oC for 4 days and the content of the flask was filtered through four layers of cheese cloth. The metabolic liquor was centrifuged at 5000 rpm for 20 minutes and the supernatant obtained was assayed for extracellular enzyme activity. The mycelial mass was washed with distilled water and dried to a constant weight. The partially dried mycelia were crushed in a grinder with small amount of distilled water for the study of intracellular enzymes.

Enzyme assay procedure:- The enzyme was assessed at a pH 5.0 to 5.2 by making use of following procedures:- (i) Determination of reducing sugars by cupric ion reduction method of Somogyi Nelson (6). (ii) Depolymerisation of substrate by measuring fall in viscosity. The percent loss in efflux time

(PLE) was calculated according to the formula:- PLE = Eh - Et x100 Eh - Ew where, Eh stands for efflux time of cellulose solution containing culture filtrate autoclaved at

15lbs/sq. inch for 30 minutes, Et stands for efflux time of cellulose solution containing active culture filtrate after incubation for time t and Ew stands for efflux time of distilled water.

(iii) Identification of biodegradation

products by TLC on silica gel plates using acetone: water: chloroform: methanol ( 8: 0.5: 1:1, vvvv ) as the solvent system.

Preparation of stock solution of fungicides – A stock solution 1.5x10-2 M pentachlorophenol (PCP) was prepared in ethanol and that of m-dinitrobenzene (MDB) in iso-propanol and subsequent dilution with distilled water gives a solution of desired concentration.

In order to carry out inhibition studies, the enzyme was first purified and characterized. The purification studies were carried out with the extracellular cellulase enzymes. Employing a combined procedure of dialysis of culture filtrate, ammonium sulphate salt precipitation, ion-exchange chromatography on DEAE Sephadex A-50 and gel filtration through Sephadex G-100 and Sephadex G-200, the enzyme was purified to 32.8 folds. All

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operations were carried out at 4oC in the cold room. Sephadex G-200 fraction of the purified enzyme was lyophilized for kinetic studies.

RESULTS AND DISCUSSIONS A .niger showed maximum enzyme production in Czapek’s medium. Vitamins like biotin, calcium pentothenate, riboflavin and thiamine-HCl could enhance the enzyme activity to a great extent but low activity was observed with nitrogen sources, like asparagine, and .sodium nitrate. All cellulase components of A .fumigatus gave maximum production on the 12th day of growth in basal medium containing cellulose as sole carbon source and a combination of ammonium sulphate and ammonium dihydrogen phosphate as nitrogen source (8).The maximum yields of cellulases from A. terreus have been reported after 120h of incubation (3). The amount of reducing sugars formed at the end of enzymic hydrolysis of carboxy methyl cellulose (CMC) and the fall in viscosity due to depolymerisation of CMC by the enzymes in absence of fungicides were estimated. (Table 1). In presence of effective dose of the two fungicides, no active enzymes have been found to be elaborated. It is thus concluded that these fungicides are effective in totally controlling the elaboration of cellulolytic enzyme. Table 1 indicates that the peak activity of intracellular and extracellular enzymes is recorded on 4th day of incubation.

The extracellular enzymes have been found to be more active in hydrolyzing as well as depolymerising the substrate than the intracellular enzymes.Various factors relevant for improvement of enzymatic hydrolysis of lignocellulosic material saw dust have been focused by Acharya et al, 2008.(1). From the studies on effect of two fungicides, it is evident that 1.5 x 10-3 M solution of MDB is effective in bringing about 84.5% inhibition while 1.5 x 10-3 M solution of PCP is effective in bringing about 76% inhibition of enzyme activity of A. niger. Any further increase in concentration of either of the fungicides did not cause any appreciable increase in inhibition of enzyme activity. (Table2). Although the action of m-dinitrobenzene in controlling the depolymerisation of CMC is faster, yet it does not reach to completion irrespective of the concentration of the fungicide used. MDB is, therefore, a stronger inhibitor than PCP in hydrolyzing the substrate while PCP controls the depolymerisation of CMC more effectively than MDB. The inhibiting characters of PCP against A. niger have been reported by Chatterjee et al, 1961 (4). Parachloromercuric benzoate inhibited cellobiase activity of A. niger by 68% while Ethylene diamine tetra acetic acid (EDTA) inhibited the cellulase activity of A. niger by 15%. (7).The cellulase enzyme from tomato fruits deteriorated by A. flavus was found to be inhibited by EDTA, 2,4-dinitrophenol and mercuric chloride(2).

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The maximum activity of the enzyme was observed at pH 5.0. The temperature optimum of the purified enzyme was determined and it was found that the enzyme exhibited its full activity at 55o C. The preincubation at 55o C showed no appreciable loss in activity but after preincubation at 60o C , there was a small fall in activity and at 70o C a sharp fall in activity of carboxymethyl cellulase enzyme was observed. The half life (t1/2) of the enzyme at 70o C was assessed 42 minutes at optimum pH. It was further observed that the enzyme was completely inactivated when incubated for 15 minutes at 80o C. The effect of varying substrate concentration ,S, on activity of purified enzyme was studied.

The initial velocity ,V, was estimated by measuring the reducing sugar released. Then a double reciprocal plot of Lineweaver and Burk was formed by plotting 1/V against 1/S , when Vmax was found as 6.65 µM/sec. and Km as 13.3mM. Dixon plots for both the inhibitors gave inhibition constant (Ki) to be equal to 19.2 mM for MDB and 22.6 mM for PCP indicating thereby that MDB is a more potent inhibitor than PCP. The Energy of Activation was measured at eight different temperatures. At each temperature the value of Vmax was measured. The logarithm of Vmax was plotted against 1/T to obtain the Arrhenius Plot as a straight line. The slope of this line was found to be 2.16x 103 and Ea to be 41.34 KJ/mole.

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Table 1 Isolation of extracellular and intracellular enzymes of Aspergillus niger in absence of

fungicides

Table 2 Enzyme activity of Aspergillus niger in presence and absence of different concentrations of

fungicides

Fungicide Final Concentration

Enzyme Activity (µg/ml)

% Inhibition

Control Nil 710 0.0

m-Dinitrobenzene 1.5 x 10-6 520 26.8

1.5 x 10-5 400 43.7

1.5 x 10-4 290 43.7

1.5 x 10-3 110 84.5

1.5 x 10-2 100 85.0

Pentachlorophenol 1.5 x 10-6 550 22.5

1.5 x 10-5 490 31.0

1.5 x 10-4 330 53.5

1.5 x 10-3 170 76.0

1.5 x 10-2 170 76.0

REFERENCE (1) Acharya,P.B.; Acharya D.K. and Modi

H.A. African J. of Biotechnol. 7(2), 4147-4152, 2008.

(2) Ajayi,A.A. ;Adejuwon,A.O. ; Awojobi, O.K. and Olutiola, P.O. Pakistan Journal of Nutrition. 6(2); 198-200, 2007.

(3) Ali, S.; Syed, A.; Sarkar, R.I.: Alam, R. World J. Microbiol. Biotechnol.7(1), 62 ,1991.

(4) Chatterjee, M.G.; Ranganathan, S.K.; Saxena, B.B.L. and Sengupta, S.R. Defence Sci. J. 11(3), 40, 1961.

(5) Chellapandi,P. and Jani, H.M. Bra. J. Microbiol.,39; 122-127, 2008.

(6) Nelson,N. J. Biol. Chem. ;153, 375, 1944. (7) Singh, A.; Agarwal,A.K.; Abidi, A.B.;

Darmwal,N.S. Appl. Microbiol. Biotechnol. 34(3), 356, 1990.

(8) Trivedi,L.S.: Rao, K.K. Ind. J. Exp. Biol. 17(7), 671, 1979.

Extracellular enzymes Intracellular enzymes

Growth Period (days)

Reducing sugars formed (µg/ml)

% fall in viscosity

Reducing sugars formed (µg/ml)

% fall in viscosity

2 - - - -

4 715 85 665 53

6 680 80 665 50

8 500 72 510 44

10 425 48 405 38

12 400 35 385 26