Production, Purification and Characterization of Cellulases from Trichoderma harzianum in Submerged Condition and their Applications

Abstract

Cellulase constitute the third largest group of enzymes employed in the industry for the processing of food, textile, detergents, paper and pulp and conversion of agricultural biomass into bio fuel and other useful products. The present study therefore was focused on production of cellulases from Trichoderma harzianum for employing in various applications. Trichoderma harzianum after qualitative screening on carboxymethyl cellulose agar and cellulose Azure was utilized for the production of endoglucanase, exoglucanase and β- glucosidase. Mandel`s mineral salt medium was found to be suitable for the production of cellulases in submerged culture at 120rpm. Significant improvements in specific activities of cellulases were recorded in the Mandel’s salt medium supplemented with 3% wheat bran and 0. 06% lactose as an inducer and addition of Tween 80 further facilitated cellulase production. Maximum production of all the enzymes was achieved between 72 -96 hours of fermentation in most of the production parameter notable activities were achieved at 96 hours. The enzyme production was favorable at temperature of 25°C and 30°C with optimum production reported at 30°C with significant specific activities of endoglucanase (137.3U/mg), exoglucanase (80.63 U/mg) and β-glucosidase (106.88 U/mg). pH 5.0 brought significant production of endoglucanase (139.26 U/mg), exoglucanase(90.12 U/mg) and β-glucosidase (109.88U/mg). Purification was carried out on Sephadex G-100, which revealed two distinct peaks of enzyme activity. Lyophilized fractions with 425.7% yield for endoglucanase, 464. 6% exoglucanase and 468% β-glucosidase were subjected to SDS-PAGE for the determination of molecular weights that resulted in separation of protein into five bands of 95KDa, 75 KDa, 65KDa, 60KDa and 20KDa. Characterization of crude and purified cellulases indicated maximum residual activities of crude cellulases between 35-55°C with maximum activities attained at 45°C. The temperature optima of purified cellulases shifted from 45°C to 50°C for endoglucanse and exoglucanase and to 55°C for β-glucosidase. Study of residual activity of crude and purified cellulases in varying pH revealed that the enzymes have a broad pH range both crude and purified fractions were most active at pH5- _______________________________________________________________________________ Production, Purification & Characterization of Cellulases from Trichoderma harzianum in Sub merged Condition & their Applications. 6. 5 with maximum residual activities retained at pH 5.5. Metal ions like Mn2+, Zn2+, Ca2+, Mg2+, K, and Na had stimulatory effect on the activity of crude as well as purified cellulases Fe2+ ion neither inhibited nor had a stimulatory effect. Lead, Hg2+ and Cu2+ had maximum inhibitory effect. Co2+ inhibited endoglucanase and exoglucanase activity while a slight inhibition was reported in case of β-glucosidase. Enzyme activities of purified enzymes were stimulated by mercaptoethanol. SDS and EDTA had a slight inhibitory effect. Crude enzyme were supplemented in a high fiber formulated poultry feed. Seven dietary treatments comprising of four dietary doses of cellulases only, two dietary mixed doses with cellulases and proteases and a dietary dose without enzyme supplementation that was kept as a control were fed to birds over a period o f 43 days. Enzyme supplementation resulted in increase in average weight gain of all the treatments when compared with control. 156g more weight than control group (A) was recorded in group E (16ml cellulase /kg feed) on 43rd day. A decrease in feed conversion ratio was recorded in all the treatment groups that were linear with increasing enzyme doses. With increasing cellulase dose a decrease in viscosity was noted. In vitro maximum viscosity (1.29 cP) was recorded in control group (A) and minimum was in group E (0.712 cP). Similar results were reported for In vivo viscosity studies of digesta from live birds with maximum figure (1. 089 cP) in control group (A) and maximum reduction was attained in group E (0.521). Dry matter digestibility in control group (80.58%) was less than in all treatment groups with maximum reported in group G (91.08%). Deinking of different type of waste paper resulted in increase in color units of all treated pulps over their respective controls. An average of 2% increase in brightness index of all treated pulps was achieved. Image analysis revealed reduction of residual ink (%) in all the enzyme treated hand sheets. The crude enzyme was found to be stable in all five commercial detergents used, with 90% of residual activity retained after 1 hour of incubation at 40 °C in detergent A. More than 40% residual activity was retained in rest of the detergents after one hour. The crude enzyme was active in removing/fading different type of stains and along with detergent the cleaning power was enhanced further. The crude preparation of cellulases was also successful in biostoning of denim bringing out an even faded look of the garment with an additional advantage of negligible back staining.