Isolation and functional charecteristics of xylanases and cellulases of thermoascus aurantiacus

Abstract

ISOLATION AND FUNCTIONAL CHARACTERISTICS OF XYLANASES AND CELLULASES OF THERMOASCUS AURANTIACUS” SUBMITTED BY KIRAN KHANDKE FOR THE Ph.D. DEGREE OF THE INDIAN INSTITUTE OF SCIENCE, BANGALORE - 560 012 The thesis deals with the purification of xylanolytic and cellulolytic enzymes from a thermophilic fungus, Thermoascus aurantiacus, and the study of their physicochemical properties and their functional properties in the hydrolysis of larchwood xylan. Plant cell wall constituents, cellulose and xylan, are among the most abundant and renewable organic compounds on earth. The study of their biodegradation to the constituent monomeric saccharides has assumed considerable importance in recent times because of the potential use of the monomers for a variety of industrial applications. Bioconversion of cellulose and xylan to the monomers is brought about by the action of the enzymes cellulases and xylanases. These enzymes have been obtained mainly from mesophilic microorganisms. Enzymes from thermophilic fungi have not been studied to that extent. In the present investigation, which aims at the characterization of these enzymes and the study of the mode of action of xylanases from a fungal source, a search was made for a thermophilic fungus which produces appreciable amounts of thermostable cellulolytic and xylanolytic enzymes. Several soil samples were screened for the isolation of thermophilic cellulolytic fungi. The isolated fungi were screened for their ability to produce extracellular cellulase. One of them was chosen for further study as it elaborated large amounts of cellulase into the culture media. This fungus was identified by various criteria as Thermoascus aurantiacus. Its growth conditions to obtain high cellulase activity were standardized. The pattern of production of xylanase and cellulases under different growth conditions was analysed. The utilization of cellulose from different cellulosic materials by this fungus was also studied. A procedure was evolved to fractionate the cellulases and xylanases on a single column of DEAE Sephadex A 50 using four elution buffers of different pH and ionic strengths. Further purification was done using gel filtration and adsorption chromatography. An endoxylanase, an endocellulase and a glucosidase were obtained in the homogeneous state, whereas an exocellulase and an D glucosiduronase were obtained in a highly purified state. Enzymes exhibiting cellulase activity consisted of an endocellulase, an exocellulase and a glucosidase, while the xylan degrading system consisted of an endoxylanase, the glucosidase (which exhibited xylosidase activity) and a debranching enzyme, D glucosiduronase. An enzyme exhibiting D glucosiduronase activity is reported for the first time. This enzyme specifically cleaves off the (1 2) linked 4 O methyl D glucuronic acid side groups of larchwood xylan and the acidic oligosaccharides derived from it, leaving the (1 4) linked xylan backbone intact. The endoxylanase was crystallized. Physicochemical and kinetic properties of four enzymes were studied. They were found to be active and stable at high temperatures, especially the endoxylanase and the endocellulase, which retain complete activity even at temperatures up to 70°C for at least 8 hours. The temperature optima for endocellulase, glucosidase, endoxylanase and D glucosiduronase were 76°C, 71°C, 63°C and 65°C respectively, and they were optimally active at pH 2.9, 4.2, 5.2 and 4.5. The sedimentation coefficient (s ,w) of endoxylanase was 3.0 while for endocellulase it was 2.7. The carbohydrate contents of the enzymes were: endoxylanase - 1.0%, endocellulase - 1.7%, glucosidase - 15%, and exocellulase - 5.2%. The molecular weights as determined by SDS gel electrophoresis were 31,800; 32,200; 58,000; 98,000; and 118,000 daltons for endoxylanase, endocellulase, exocellulase, glucosidase and D glucosiduronase respectively. The amino acid composition of endoxylanase, endocellulase and glucosidase showed that all three were rich in Asp/Asn and poor in sulfur containing amino acids. The kinetic constants for different substrates were also determined. Larchwood xylan was found to be hydrolysed in a random manner by the endoxylanase to give a series of neutral and acidic oligosaccharides, the proportion of each varying with the extent of hydrolysis. The partial hydrolysis products were purified and identified as neutral saccharides consisting of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, and acidic oligosaccharides with a backbone of 3 to 7 xylose residues, each having a 4 O methyl glucuronic acid side group on one of the xylose residues. Complete hydrolysis of xylan by xylanase yielded xylose, xylobiose and an acidic tetrasaccharide whose structure was determined as: (1 4) (1 4) xylose - xylose - xylose (1 2) 4 O methyl glucuronic acid The neutral xylo oligosaccharides were reduced by NaBH to the corresponding alcohols and were used to determine the mode of action of endoxylanase and glucosidase. Glucosidase was found to cleave one xylose residue at a time from the non reducing end, while endoxylanase cleaved the glycosidic linkage at the central region of the oligosaccharide chain. It was found that endoxylanase catalysed the hydrolysis of xylo oligosaccharides at different rates depending on the degree of polymerization, the rate increasing with increasing polymer length. In contrast, glucosidase hydrolysed all the xylo oligosaccharides at nearly equal rates. Analysis of the products of hydrolysis of acidic oligosaccharides (derived from partial hydrolysis of xylan) by endoxylanase indicated that its action is blocked one xylose residue on either side of the xylose carrying the acidic side group. In contrast, glucosidase action was blocked two xylose residues from the substituted xylose, toward the non reducing end. Kinetics of hydrolysis by D glucosiduronase of the acidic oligosaccharides and xylan indicated that these are hydrolysed at comparable rates, showing that the position of the side group is not important for D glucosiduronase action. Hydrolysis of the (1 4) linkage between xylose residues was not detected in any xylo oligosaccharide tested using D glucosiduronase. To conclude, endoxylanase, glucosidase and D glucosiduronase of T. aurantiacus were found to constitute the complete enzyme system for the hydrolysis of larchwood xylan to its constituent monomers.