International Journal of Pharma and Bio Sciences
RESEARCH ARTICLE
ARTICALTICLE
BIO CHEMISTRY
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.
SAXENA ARTI
Deptt. of Chemistry , A.N.D.N.N.M.College, Kanpur.
*Corresponding author
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.
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KEYWORDS
Biodegradation, fungicides, inhibitive
INTRODUCTION
Cellulase enzymes from various microorganisms, 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
enzymes elaborated by the fungus.
cellulase
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, ionexchange 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 thiamineHCl 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
Extracellular enzymes
Intracellular enzymes
Growth Period
(days)
Reducing
sugars formed
(µg/ml)
Reducing
sugars formed
(µg/ml)
2
4
6
8
10
12
715
680
500
425
400
% fall in
viscosity
85
80
72
48
35
% fall in
viscosity
665
665
510
405
385
53
50
44
38
26
Table 2
Enzyme activity of Aspergillus niger in presence and absence of different concentrations of
fungicides
Fungicide
Final
Concentration
Control
m-Dinitrobenzene
Pentachlorophenol
Enzyme Activity
(µg/ml)
Nil
1.5 x 10-6
1.5 x 10-5
1.5 x 10-4
1.5 x 10-3
1.5 x 10-2
1.5 x 10-6
1.5 x 10-5
1.5 x 10-4
1.5 x 10-3
1.5 x 10-2
% Inhibition
710
520
400
290
110
100
550
490
330
170
170
0.0
26.8
43.7
43.7
84.5
85.0
22.5
31.0
53.5
76.0
76.0
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