REGULATION OF THE CELLULASE ENZYME SYSTEM IN THE THERMOPHILIC FUNGUS THERMOASCUS AURANTIACUS
Abstract
Thermoascus aurantiacus is a thermophilic cellulolytic fungus capable of good growth and enzyme production at 50C. T. aurantiacus produces cellulase enzyme when grown on a wide variety of cellulosic as well as non-cellulosic substrates. The enzyme system was stable at 70C for at least 24 h. In time course studies on 1.0% glucose, CM-cellulase production began when the glucose concentration was reduced to a non-repressive level (ca. 0.2 gm glucose/100 ml). (beta)-Glucosidase and (alpha)-amylase, enzymes considered constitutive in other organisms, were not produced until much later when autolysis became evident. Culture aeration greatly affected the rates of glucose uptake and cell growth rate as well as the time and amount of enzyme produced. These parameters increased as a function of increased aeration. The cellulase enzyme components were identified on non-denaturing (native) polyacrylamide gels by direct assay procedures. These protein bands were then cut out and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes have been found to have molecular weights of: (beta)-glucosidase, 85.0 kDa; exoglucanase, 40.0 kDa; and endoglucanase, 32.0 kDa. (alpha)-Amylase, a constitutively produced enzyme in many fungi was found to have a molecular weight of 52.0 kDa in T. aurantiacus. SDS-polyacrylamide gels of time course fermentation filtrates indicated very good correlation between protein production and the detection of enzyme activity. The ability of substrates to act as repressors or inducers was studied. Only cellulosic materials were found to act as inducers. Neither sophorose nor gentiobiose, strong inducers in Trichoderma reesei, caused induction in T. aurantiacus, Glucose was repressive at high concentrations but CM-cellulase was produced at a constitutive level after the glucose concentrations were reduced to non-repressive levels. Sodium acetate appeared to cause an immediate decrease in the rate of CM-cellulase production along with enzyme inactivation. It has not been determined if these effects are due to the ability of the sodium acetate to act as a cellulase repressor or its ability to inhibit activity while not affecting synthesis.
Degree
Ph.D.
Subject Area
Microbiology
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