Production, p urification 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, tem perature , moisture content, incubation period) and effect of co - inducers on production of cellulase (FPase, CMCase, and cellobiose ) produced by A. flavus and A. japonicum isol ated 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 endogl ucanase (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 s ugarcane baggase and wheat straw . The hig hest 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 cellobia se 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 4 5min of incubation at 50° C. The endoglucanase activity stimulated 66% by Co 2+ however, Hg 2+ competitively inhibited CMCase with a K i of 2μM. The value of K m and V max 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 N CBI accession no. KF716164 (A. flavus ) and KF716165 ( A. japonicum ) . Key words: Bioconversion, CMCase, Cellobiase, FPase , SSF, Saccharification .
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DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY
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