EXTRACTION PURIFICATION AND CHARACTERIZATION OF
CELLULASE OF CELLULASE ENZYME COMPLEX FROM
TRICHODERMA T5
Ansari Nayab Taher*
Department of Zoology, Sir Sayyed College, Aurangabad.
ABSTRACT
The cellulase enzyme complex was extracted from Trichoderma T5.
The enzyme was purified using column chromatography. The effect of
different parameters on the activity of enzyme was determined, it was
found that the enzyme had optimum temperature of 30° C, the
optimum pH was 5.5, the optimum substrate concentration was found
to be 2.0% W/V of carboxyl methyl cellulose also it was found that
time of incubation influences the enzyme activity it was found that the
optimum time of incubation was 96 hours after which the activity
decreases. Thus, the cellulase enzyme complex was extracted from
Trichoderma T5 and further purified by column chromatography and it proves to be a
efficient method of purification of enzyme complex and the activity of enzyme is effected by
different parameters.
KEYWORDS: Purification, Cellulase enzyme, Trichoderma T5.
INTRODUCTION
Enzymes are found in complex mixtures, usually in the cells which perhaps contain many
different types of enzymes and in order to study a given enzyme properly it must be purified.
The conversion of cellulose into glucose is brought about by cellulase enzyme complex.
Targonski (1995) the cellulase enzyme complex comprises of endo – β – 1 – 4 glucanase, exo
– β – 1 – 4 and – glucosidase. The mode of action of each of these being.
Endo – β – 1 – 4 glucanase: - It cleaves the cellulose chain at random intervals yielding cello
oligosaccharides and glucose.
Volume 8, Issue 13, 988-993. Research Article ISSN 2277– 7105
*Corresponding Author
Ansari Nayab Taher
Department of Zoology, Sir
Sayyed College, Aurangabad. Article Received on
06 Oct. 2019,
Revised on 27 Oct. 2019, Accepted on 17 Nov. 2019
Exo – β – 1 – 4 Glucanase: - It is an exo enzyme which hydrolyses cellulose chain and
attacks on non reducing end of cellulose and removes cellobiose.
β – Glucosidase:- It catalyzes the hydrolyses of cellobiose and cellodextrins to glucose.
The advantage of enzymatic bioconversions are that the pH and temperature conditions are
less severe than those of chemical processes and very pure products are obtained without
secondary byproducts. The most active cellulases are derived from Trichoderma. reesei,
T.lignorum, T.koningii, T.pseudokoningi. Enzyme activities are known to be influenced by
temperature, pH, time of incubation, substrate concentration, inhibitors and activators.
Tikhomirov. Et. Al., (1992) have reported highly thermostable cellulase complex from the
bacteria Anaerocellum thermophillum which can tolerate temperatures upto 68 – 75° C.
although some cellulases have optimum activity even under mesophilic conditions. pH do
affect the activity of enzyme. Levin et al. (1997) has found that in general most cellulases are
In the present investigation an attempt was made to extract and purify the cellulase enzyme
complex from Trichoderma T5 using column chromatography and to study the effect of
temperature, pH, time of incubation and substrate concentration on the activity of cellulase
enzyme.
MATERIAL AND METHODS Extraction
The fungus Trichoderma T5 was inoculated in 2 liters of cellulose minerals salt media
containing 0.2% W/V carboxymethyl cellulose. The culture was grown at 30° C for 4 weeks.
After 4 weeks the culture was filtered and the supernatant was centrifuged at 8000 rpm for 20
minutes. The enzyme was precipitated by 8% ammonium sulphate. The resulting precipitate
was dissolved in 50 ml of sodium citrate buffer of pH 5.5, the enzyme was recentrifuged and
dialyzed against sodium citrate buffer of pH 5.5 for 24 hours and this was treated at crude
enzyme.
Purification of Cellulose Enzyme Complex
500 grams of DEAE sephadex A – 25 was allowed swell overnight in distilled water, it was
then washed in Buchner’s funnel with 0.5 M Hydrochloric acid and 0.5 M sodium hydroxide.
and the DEAE sephadex A – 25 was poured into a glass column having cotton plug at the
was mixed in 15 grams of sephadex and continuously stirred till free flowing and applied to
the top of the column. Elution was carried in Isocratic or ingradient mode as required. Several
fractions of 2 ml were collected and scanned in Uv – spectrophotometer at 260 and 280 nm.
The fractions showing – max at 280 nm were selected and used as pure enzyme (Table –
1).
Table 1: Spectrophotometer Scanning of The Fractions. Fraction no. 260 mm 280 mm
1 - -
2 - -
3 - -
4 - -
5 - -
6 0.131 140
7 0.138 003
8 0.123 001
9 0.132 007
10 0.111 123
11 0.004 162
12 0.002 008
13 0.134 -
14 0.111 174
15 0.120 0.006
16 0.162 0.031
17 0.071 0.02
18 0.009 0.180
19 0.110 0.180
20 - 0.121
21 - -
22 - 0.003
23 - 0.561
24 - 0.123
25 - 0.111
26 - 0.100
27 - 0.505
28 - 0.480
29 - 0.211
30 - 0.192
31 - 0.098
32 - 0.214
33 - 0.121
Cellulase Assay Using Saccharification
All the fractions having max at λ 280 nm were assayed for its cellulolytic activity by
saccharification process as given by Sumner (1935).
Effect of Different Parameters on Enzyme Activity
Following the method of Sumner (1935) the pure enzyme cellulase was studied for
determining the effect of different parameters. The temperature selected was 10 – 50° C for
pH the effect of 3.5 – 8.0 pH was determined. The substrate concentration was used was
0.5% W/V to 3.0% W/V. The effect of time of incubation of the activity of cellulase enzyme
was carried out for 12, 24, 48, 96 and 108 hours.
RESULTS AND DISCUSSIONS
The cellulase enzyme complex was extracted and purified from Trichoderma T5. It suggests
that the fungi are efficiently cellulolytic in nature, Mohagheghi et al. (1992) reported
degradation of cellulose using enzymes of T.reesei and the had high saccharification ability.
It was found that there were many fractions having λ max at 280 nm. It appears that still
much work has to be done on purification of individual components of the enzyme. With the
help of column chromatography it just possible to purify the whole cellulase enzyme
complex, but still a right method has to be invented which will purify the subcomponents of
cellulase enzyme complex. Recently electrophoretic techniques have been used for the
purification of different endoglucanases, exoglycanases and λ-glucosidase. Nicoli et al.
(1995).
The cellulase enzyme tested showed the optimum activity at 30°C (fig.1). Fadel et al (1996)
investigated into cellulase enzyme from Penicillium steckii and A.niger F-92 respectively
which had the optimum activity at 30°C. The optimum pH was found to be 5.5 (fig.2). It does
indicates the enzyme is well capable of tolerating acidic conditions. The optimum substrate
concentration was found to be 2.0% carboxy methyl cellulose W/V (Fig 3). However,
increasing amount of substrate concentration may not increase the activity of enzyme.
Khanok Ratanakhanokchai (2000) has determined the effect of cultural condition on cellulase
by thermophilic Bacteroids sp. Strain P-1 and found that a concentration of 30 grams per liter
of soyabean meal as optimum substrate concentration. The optimum period of incubation was
96 hours (Fig. 4). The incubation time does affect the enzymatic activity attributing to the
complete degradation is more thus greater is the complexity of cellulose more is the time
required for the degradation.
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