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The agricultural organic wastes pose major environmental issues and their inappropriate disposal is a major cause of pollution while these as cheap lignocellulosic resources can be utilized for many value added products such as enzymes. The purpose of the present study was to utilize wheat bran as a sole substrate for enhanced cellulase production under submerged fermentation (SMF) or solid state fermentation (SSF) and optimize the various process variables involved in the selected fermentation type. In order to achieve high titer of cellulase, a central composite design (CCD) was constructed and performed for optimization of SSF with five process variables at five coded levels. A 25 full factorial design was constructed leading to a set of 50 experiments that were performed in triplicates. The key variables namely incubation time, temperature, wheat bran and tap water ratio, pH and inoculum size were evaluated. The optimization of the process variables resulted in 1.14 IU/ml of cellulase activity from Bacillus subtilis PJK6 under SSF using wheat bran as a sole organic substrate with tap water. The optimum conditions included incubation time- 72 h, temperature- 45oC, pH- 6, inoculum size- 14% with 1:4 as wheat bran and tap water ratio. The production of cellulase using only moistened wheat bran was demonstrated and found to be significantly controlled by incubation time and temperature while pH showed the least effect. The economic production of valuable and useful enzyme using agricultural residue was achieved at moderate conditions from a GRAS microbe that can benefit the industry as well as the environment.
The goal of this work was to clone, express, characterize and assemble a set of soluble thermostablecellulases capable of significantly degrading cellulose. We successfully cloned, expressed, and purified eleven Clostridium thermocellum (Cthe) cellulases and eight Acidothermuscellulolyticus(Acel) cellulases. The performance of the nineteen enzymes was evaluated on crystalline (filter paper) and amorphous (PASC) cellulose. Hydrolysis products generated from these two substrates were converted to glucose using beta-glucosidase and the glucose formed was determined enzymatically. Ten of the eleven Cthe enzymes were highly active on amorphous cellulose. The individual enzymes all produced <10% reducing sugar equivalents from filter paper. Combinations of Cthe cellulases gave higher conversions, with the combination of CelE, CelI, CelG, and CelK converting 34% of the crystalline cellulose. All eight Acel cellulases showed endo-cellulase activity and were highly active on PASC. Only Acel_0615 produced more than 10% reducing sugar equivalents from filter paper, and a combination of six Acel cellulases produced 32% conversion. Acel_0617, a GH48 exo-cellulase, and Acel_0619, a GH12 endo-cellulase, synergistically stimulated cellulose degradation by the combination of Cthe cellulases to almost 80%. Addition of both Acel enzymes to the Cthe enzyme mix did not further stimulate hydrolysis. Cthe CelG and CelI stimulated cellulose degradation by the combination of Acel cellulases to 66%.
Trichoderma reeseiβ-glucosidase (Bgl1) is one of four enzymes demonstrated to act synergistically to degrade cellulose both in vitro and in vivo. Our work attempted to better understand the substrate specificity and potential biotechnological applications of Bgl1. T. reesei Bgl1H cleaves over 80% of the β-(1-4) and β-(1-3) linkages in β-glucan and 14% of the β-(1-4) linkages in amorphous cellulose, significantly more than any tested bacterial β-glucosidase. Bgl1H cleaves 50% of the β-(1-4) linkages in xyloglucan when supplemented with cellulase and α-xyloside. Approximately 20% conversion to glucose was obtained from insoluble β-(1,3)-linked curdlan using only Bgl1H; addition of a curdlanase resulted in conversion of approximately 70% of the curdlan to glucose. Bgl1H also produces xylose from xylooligosaccharides and debranched xylans. For both glucans and xylans, the relative rates of hydrolysis increase with increasing polysaccharide chain lengths. Bgl1H is able to partially degrade β-glucan in a variety of grain components; addition of endo-acting enzymes improved the enzyme’s performance on these grain components. The ability of this enzyme to produce monosaccharides from undigestible polysaccharides suggest it may have potential in improving utilization of carbohydrates in animal feed, fermentations, and other biotechnological applications.