Production, purification and characterization of cellulase from Aspergillus species
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Date
2017
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DEPARTMENT OF BIOCHEMISTRY AND BIOCHEMICAL ENGINEERING, JACOB SCHOOL OF BIOTECHNOLOGY AND BIOENGINEERING, SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES, ALLAHABAD- 211007
Abstract
Optimization parameters (pH, temperature, moisture content, incubation period) and effect
of co-inducers on production of cellulase (FPase, CMCase, and cellobiose) produced by
A. flavus and A. japonicum isolated from soils of Allahabad, U.P. via solid state fermentation
(SSF) using sugarcane bagasse, wheat straw and paper waste as substrates was studied. In
crude enzyme extracts both strains exhibited higher endoglucanase (CMCase) activity as
compared to cellobiase and total cellulase activity (FPase). The specific activities of the three
enzymes also followed a similar pattern i.e. highest in paper waste as compared to the
sugarcane baggase and wheat straw. The highest production of cellulase was obtained with A.
flavus when 5.0g paper waste in 1:3 basal medium ratio growth medium (30mL); 1.0% (w/v)
yeast extract and cellulose as co-inducers was used at 30°C, pH 4.5, 96 hrs of SSF CMCase
1150.81 ± 6.26 IU/L cellobiase 670.44±5.8IU/L and FPase 598.53±5.56 IU/L). The
optimized conditions were used to extract and purify CMCase by a three step procedure, the
purification factor was 462.5 after gel filtration yield 4% Molecular weight of CMCase was
found to be ~23 KDa as determined by SDS-PAGE. The purified endoglucanase retained
90%of its maximum activity after 45min of incubation at 50°C. The endoglucanase activity
stimulated 66% by Co2+ however, Hg2+ competitively inhibited CMCase with a Ki of 2μM.
The value of Km and Vmax were increased 16.66% by 33.33% after purification. The
efficiency of crude cellulase produced by A. flavus and A. japonicum in bioconversion was
also compared. With purified enzyme the degree of saccharification was lower than crude
enzyme for all the substrates. Though, paper waste was saccharified the most among all
substrates. Molecular characterization of the two Aspegillus species revealed two novel
strains and were assigned NCBI accession no. KF716164 (A. flavus) and KF716165 (A.
japonicum
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