Kinetic study of partially purified cellulase enzyme produced by Trichoderma viride FCBP-142 and its hyperactive mutants

• Published in: Microbiology (New York) 2011-06, Vol.80 (3), p.363-371
• Main Authors: Shafique, Shazia, Shafique, Sobiya
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Experimental Articles; Life Sciences; Medical Microbiology; Microbiology; Trichoderma viride
• ISSN: 0026-2617; 1608-3237
• DOI: 10.1134/S0026261711020135

Cellulases are the enzymes that cleave β-1,4 linkages of cellulose, and carbohydrate that is main part of plants’ cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50°C with E a for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol −1 and temperature quotient of 1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50°C for about 60 min but rapid denaturation occurred above 55°C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKa 1 and pKa 2 . The pKa1 of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa 2 moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant K m 0.6, 0.5 and 0.28 mg mL −1 and V max value of 8.33, 10 and 9.09 Units mL −1 for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.