| dc.description.abstract | ISOLATION AND CHARACTERIZATION OF POLYSACCHARIDE DEGRADING ENZYMES OF Chaetomium thermophile var. coprophile”
SUBMITTED BY: RAMESH KUMAR GANJU
FOR THE Ph.D. DEGREE OF
INDIAN INSTITUTE OF SCIENCE, BANGALORE – 560012
The thesis deals with the purification of cellulolytic and xylanolytic enzymes from a thermophilic fungus, Chaetomium thermophile var. coprophile, and the study of their physicochemical and functional properties.
Cellulose and xylan, the structural components of the plant cell wall, are the world’s most abundant and renewable organic polymers and therefore represent important raw materials for the production of valuable chemicals. The first step in this process is the enzymatic degradation of these polysaccharides into their monomeric saccharides by cellulases and xylanases. Considerable work has been carried out on these enzymes from mesophilic sources; however, enzymes from thermophilic fungi have not been investigated extensively. The present study deals with the cellulases and xylanases from a thermophilic fungus.
Various strains of seven species of thermophilic fungi were isolated from different soil samples. The isolated fungi were screened for their ability to produce extracellular cellulase. One of the fungi identified as Chaetomium thermophile var. coprophile, based on several criteria, was chosen for detailed study due to its ability to produce comparatively larger quantities of enzymes. A suitable medium for growth and cellulase production was established. The pattern of cellulase production under different growth conditions was analysed. The saccharification ability of the culture filtrate toward different cellulosic materials was also determined.
An analysis of the total proteins from the culture filtrate by DEAE–Sephadex A 50 chromatography revealed the presence of at least two glucosidases, five xylanases, and four each of exocellulases and endocellulases. Using ion exchange and gel filtration chromatography, one glucosidase, one endocellulase, two exocellulases, and two xylanases were isolated in homogeneous form. Their physicochemical and functional characteristics were studied.
The molecular weights as determined by SDS–PAGE were:
Glucosidase: 78,000
Xylanase I: 26,000
Xylanase II: 7,000
Endocellulase: 36,000
Exocellulase I: 60,000
Exocellulase II: 40,000 daltons
The carbohydrate contents of the enzymes were:
glucosidase – 10%, exocellulase I – 17%, and exocellulase II – 22.8%.
The amino acid compositions of glucosidase, exocellulase I, exocellulase II, xylanase I, and endocellulase were determined.
The two exocellulases differed slightly in amino acid composition. Antibodies raised against exocellulase I did not cross react with exocellulase II, and vice versa, indicating immunological distinctness.
The purified enzymes were found to be active at high temperatures. The temperature optima for glucosidase, xylanase I, xylanase II, endocellulase, exocellulase I, and exocellulase II were 70 °C, 70 °C, 60 °C, 80 °C, 75 °C, and 70 °C, respectively. They were optimally active at pH 5.6, 4.8–6.4, 5.4–6.0, 6.0, 5.8, and 6.4, respectively. Exocellulase I and II were found to be more stable at 70–80 °C compared to glucosidase, endocellulase, and the xylanases. Kinetic constants for different substrates were also determined.
Studies on the activities of cellulolytic enzymes toward different polysaccharides showed the following:
Endocellulase degrades amorphous cellulose more readily than crystalline cellulose.
Exocellulases preferentially degrade crystalline cellulose, releasing cellobiose units.
Glucosidase degrades disaccharides more efficiently than oligosaccharides and is specific for diglycosides and anomers.
Mode of action studies revealed:
Both exocellulases hydrolyse cellulose by removing one cellobiose unit at a time.
Endocellulase acts randomly, producing oligosaccharides.
Glucosidase hydrolyses polysaccharides to glucose.
Xylanase II releases xylobiose, whereas xylanase I produces xylobiose, xylotriose, and small amounts of higher xylo oligosaccharides.
Studies on the degradation of cotton fibres by the cellulases showed that extensive degradation is possible if repeated applications of exocellulase I or II are made over long durations. However, a fraction of the cotton fibre remains resistant to enzymatic attack. Electron micrographs of untreated and degraded cotton fibres showed clear morphological changes due to enzymatic action.
It is evident from the present study that Chaetomium thermophile var. coprophile produces a complete multicomponent cellulolytic and xylanolytic enzyme system capable of degrading cellulose and xylan. Components of this system, particularly the exocellulases, are capable of extensively hydrolysing intact cotton fibres as well. | |
