Journal of Applied Sciences Research, 2(11): 884-889, 2006
© 2006, INSInet Publication
Triggering of β-glucosidase Production in Trichoderma viride with Nutritional
and Environmental Control
Ikram-ul-Haq, Muhammad Mohsin Javed, Zafar Siddiq and Tehmina Saleem
Institute of Industrial Biotechnology, Government College University, Lahore
Abstract: Nutritional and environmental factors have a stimulatory effect on various hydrolytic enzymes
production. The present study deals with the control of these factors for the culture of Trichoderma viride
UVNG-4 (mutant strain), in order to trigger the bio-synthesis of β-glucosidase in shake flasks. The cultural
conditions, which were optimized, include; mineral salt combinations, carbon sources, nitrogen sources, pH,
incubation time and inoculum size. Mandel and Reese mineral salts combination was better than Eggins and
Pugh with 1.0% wheat bran as a carbon source. Different types of organic and inorganic nitrogen sources were
used but soybean meal was found to be the optimum for the β-glucosidase production. The optimum
temperature, pH and incubation time were 30°C, 5.5 and 72 h, respectively. The inoculum containing 5.52×107
conidia/ml enhanced the β-glucosidase production. After the optimization of these cultural conditions,
Trichoderma viride UVNG-4 gave 19.92 U/ml/min of β-glucosidase activity.
Key words:
INTRODUCTION
MATERIALS AND METHODS
b-Glucosidases have been the subject of much recent
research due to their potential for many bio-technological
applications in food products, essential oils, pulp & paper,
jams, juices, biomass conversion and pollution control[3].
Particularly, glucose production can be achieved from the
most abundant biological macromolecule “cellulose” by
the extracellular enzyme complex (cellulases), which
comprises at least three different enzymes that together
hydrolyze cellulose to oligosaccharides and glucose[6]. Of
these, the endo-β-glucanases and cello-biohydrolases
synergistically hydrolyze cellulose into small cellooligosaccharides; mainly cellobiose. Whereas; βglucosidase hydrolyses aryl- and alkyl-glucosides,
cellobiose and cellodextrins[4,16]. Trichoderma strains have
been studied due to their ability to secrete cellulosedegrading enzymes. Much work has been carried out on
the T. viride and T. harzianum[17,7]. These strains are
improved through UV light, gamma rays and different
chemicals like; ethyl methyl sulphonate (EMS), Nmethyl-N-nitro-N-nitrosoguanidine (MNNG) and nitrous
acid (HNO2). MNNG is a potent mutagen and is being
widely used by microbiologists to obtain the organisms
capable of producing high levels of cellulases. Cultural
conditions such as carbon and nitrogen sources, ionic
concentrations, pH, incubation temperature, cultivation
time and inoculum size have fundamental role in the
growth of microorganisms and subsequent product
formation[11,7].
Microorganism: Trichoderma viride UVNG-4 was taken
from the available stock culture of Institute of Industrial
Biotechnolog, GC University, Lahore and was maintained
on (3.9 %, w/v) potato dextrose agar slants (pH 5.6).
Fermentation technique: A total volume of 1.0 ml of
conidial suspension was transferred into 250 ml
Erlenmeyer conical flasks each containing 25 ml of
fermentation medium with wheat bran as basal substrate.
After the inoculation, the flasks were incubated at
200 rpm (30ºC) in shaking incubator. After the
completion of fermentation period, fermented broth was
centrifuged at 8000×g for 10 minutes and the supernatant
was used for analytical purposes.
Analytical methods: β-Glucosidase activity was
measured after Rajoka and Malik[14] using p-nitrophenylβ-D-glucopyranoside as substrate. Protein in the cultural
broth was estimated according to the method as described
by Bradford[2]. The absorbance taken was compared with
p-nitrophenol in case of β-glucosidase and with Bovine
serum albumin in case of total protein.
RESULTS AND DISCUSSIONS
The mutant strain Trichoderma viride UVNG-4 was
selected for cultural optimization in shake flasks. Two
types (Fig. # 01) of mineral salts combination were tested.
Corresponding Author: Muhammad Mohsin Javed, Institute of Industrial Biotechnology, Government College University, Lahore
Email: mmj_bot@yahoo.com
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J. Appl. Sci. Res., 2(11): 884-889, 2006
Fig. 1: Effect of different mineral salts combination on the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates. Alphabets differ significantly
at p#0.05.
Incubation period
48 h
Initial pH
5.5
Incubation temperature
30°C
Fig. 2: Effect of different agriculture by-products on the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates. Alphabets differ significantly
at p#0.05.
Incubation period
48 h
Initial pH
5.5
Incubation temperature
30°C
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Res. J. Appl. Sci., 2(11): 884-889, 2006
Fig. 3: Effect of different concentrations of wheat bran on the production of β-glucosidase by Trichoderma viride
UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates.
Incubation period
72 h
Incubation temperature
30°C
Fig. 4: Time course study for the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates.
Incubation temperature
30°C
Mandel and Reese[10] salts combination was found to be
better than that of Eggins and Pugh[5] with 0.58 mg/ml and
8.56 U/ml/min units of protein and β-glucosidase,
respectively. It might be due to the reason that Mandel
and Reese salts combination has a balanced proportion of
macro- and micro-nutrients, required by the organism for
growth and subsequent enzyme production. Different
agricultural by-products such as wheat bran, wheat straw,
rice bran, rice straw and paper waste (Fig. # 02) were
tested supplemented with Mandel and Reese medium for
enzyme production. Of all the substrates tested, wheat
bran was found to be the best substrate for the production
of β-glucosidase, which gave maximum yield of protein
and β-glucosidase i.e., 0.3 mg/ml and 4.15 U/ml/min,
respectively. It might be due to the fact that wheat bran
provided adequate amount of nutrients like (%); proteins
(1.32), carbohydrates (69), fats (1.9), fiber (2.6), ash (1.8),
calcium (0.05), magnesium (0.17), phosphorous (0.35),
potassium (0.45), sulphur (0.12), various amino acids and
porosity for oxygen supply[1].
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Res. J. Appl. Sci., 2(11): 884-889, 2006
Fig. 5: Effect of inoculum size on the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates.
Incubation period
72 h
Incubation temperature
30°C
Fig. 6: Effect of initial pH on the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates.
Incubation period
72 h
Incubation temperature
30°C
which is required for the enzyme production and leaving
the crystalline portions, which cannot be used by the
organism for its growth and ultimate enzyme production.
Okeke and Obi[12] also produced best enzyme production
72 h after inoculation.
Production of β-glucosidase was studied by varying
the concentration of wheat bran (Fig. # 04). Different
concentrations of wheat bran were used for the enzyme
production ranging from 1.0-2.0%. Of these, 1.0%
Time course (Fig. # 03) for the enzyme production
was studied from 24-196 h. The production of enzyme
was increased with increase in fermentation period
duration and reached maximum (0.21 mg/ml for protein
and 13.29 U/ml/min for β-glucosidase), 72 h after
inoculation. Further increase in the incubation period
resulted in the decreased production of β-glucosidase.
Reduction in the enzyme production might be because of
the rapid digestion of susceptible portion of the substrate,
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Res. J. Appl. Sci., 2(11): 884-889, 2006
Fig. 7: Effect of different nitrogen sources on the production of β-glucosidase by Trichoderma viride UVNG-4
Y-error bars indicate the standard deviation among the three parallel replicates.
Incubation period
72 h
Incubation temperature
30°C
medium at 0.27% concentration replacing all nitrogen
sources from Mandel and Reese salts medium. Among all
the nitrogen sources tested; soybean meal gave maximum
production of β-glucosidase (19.92 U/ml) and protein
(1.35 mg/ml), because it was easy for the mycelium to get
nitrogen from it (Fig. # 06). But Okeke and Obi[12]
reported the more enzyme production using inorganic
nitrogen sources as compared to the organic nitrogen
sources from Arthrographis sp. They mentioned that the
use of the organic nitrogen sources could also be
advantageous than the inorganic ones, as the organic
sources are cheaper in cost and also not involved in the
competition with the active site of the enzyme. Our study
is in agreement with the work done by Kansoh and
Gammal[8]. They used organic nitrogen sources and
reported more activity of β-glucosidase than inorganic
nitrogen sources.
Effect of different inoculum sizes ranging from
1.38×107 to 8.28×107 conidia/25 ml of media was
evaluated for β-glucosidase bio-synthesis. Of all the
inoculum sizes tested; inoculum containing 5.52×107
conidia was found to be optimum, which yielded 1.11
mg/ml for protein and 16.1 U/ml/min for β-glucosidase.
As the inoculum size was increased, there was not any
rise in the production of β-glucosidase (Fig. # 07). It
might be due to the overgrowth of Trichoderma viride
UVNG-4 and the creation of anaerobic conditions.
wheat bran was optimized for maximum production of
β-glucosidase (16.6 U/ml/min) and protein (1.11 mg/ml).
Further increase in concentration of wheat bran resulted
decrease in β-glucosidase production. It might be due to
thickening of the fermentation medium, which make
hindrance in the proper agitation and aeration that resulted
decrease in air supply[13]. Other workers like Saha[15] also
selected wheat bran as the basal medium for the
production of β-glucosidase. Therefore, present study is
an agreement with the work of previous workers.
β-Glucosidase production by Trichoderma viride
UVNG-4 at different values of pH (Fig. # 05) was also
carried out. The protein (1.11 mg/ml) and β-glucosidase
(16.6 U/ml/min) were best at slightly acidic pH. It shows
that the 5.5 pH is favourable for the growth of
Trichoderma viride UVNG-4 and also suitable for the
secretion of enzyme from the fungal mycelia. There was
insufficient growth of organism in the more acidic pH and
resulting in the decreased enzyme production. The
alkaline pH does not supports fungal growth so there
was not any considerable levels of enzyme production.
These results were in accordance with the findings of
Lambert et al.[9].
In the present work, different nitrogen sources such
as urea, peptone, yeast extract, soybean meal, NaNO2,
casein, meat extract, nutrient broth, KNO3, NH4Cl and
(NH4)2SO4 were added separately to the fermentation
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Res. J. Appl. Sci., 2(11): 884-889, 2006
Zaldivar et al.[18] produced best extracellular enzymes
when submerged culture of mutant strain of Trichoderma
aureoviride in shake flask was inoculated with 5.0×106
conidia/200 ml of media in 14 days at 28°C.
9.
ACKNOWLEDGEMENT
10.
This work was financially supported by Higher
Education Commission of Pakistan under the project
No. 25 (13)/2002 DSA (P&D).
11.
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