Stability Studies Of EGX, A Multi-functional Cellulase From The Mollusca, Ampullaria Crossean

Cellulose and hemicellulose are the abundant and major constituentsfor all plants and the potential of row materials for biotechnologicalprocesses by using enzyme hydrolysis. The main use of cellulase would bein the hydrolysis of cellulosic materials in order to produce fermentablesugars for various biotechnological processes, and they were also used intextile and paper industries worldwide. It is necessary for these enzymes tofunction under extremes of pH and temperature in the industrial applicationswhere they are required. Therefore, it is desirable to understand the stabilityof cellulase enzyme and find out the best condition for its hydrolysis ofcellulosic materials. Ampullaria crossean produces a middle molecular weightmulti-function cellulase (designated as EGX) Mr 43,000. It belongs to thefamily 10 of glycosidases. EGX is a multi-function enzyme with theactivities of exo-β-1,4-xylanase, endo-β-1,4-glucanase andendo-β-1,4-glucanase. Therefore, the potential application value of EGX isconsiderable. The purification, gene cloning and subsequent expression ofthe EGX were studied in our laboratory. Despite the biotechnologicalimportance of cellulases structure-function studies of these enzymes arelimited. In the present work, we studied the effect of pH on theconformation of EGX and urea-induced denaturation by intrinsicfluorescence, ANS fluorescence, and CD measurements. At different pH values and different concentration of phosphate, theunfolding of EGX by urea was followed by residual activity of the mixture, 62吉林大学硕士学位论文 李文颖intrinsic fluorescence measurements and CD measurements. Theexperiments suggested that the concentration of phosphate had notremarkable influence upon the stability of EGX and the stability of EGXwas strongly pH-dependent. EGX at the pH 5.2 showed unusual stability to resistthe denaturation and unfolding by urea. It is important and interesting toinvestigate the structure-function relation and stability of EGX at this pH. The intrinsic fluorescence spectra of EGX suggests at pH 5.6 thetryptophan residues of the protein buried to a nonpolar environment are themost and the molecule is most compact comparing to at the other pH values.The structural alterations in the conformation of EGX were furthercorroborated by changes in ANS fluorescence. These data suggest anincrease in the hydrophobicity of the molecule except for pH 5.6. At thesepH values, the molecule becomes less compact than that at pH 5.6, andmore hydrophobic binding sites are accessible to bis-ANS. The effect of pH on the secondary structures of EGX was monitored by far-UVCD studies. The value of mean residue ellipticity at 222 nm can be used to determinethe α-helicity of a protein using a sample calculation. The estimated α-helicity ofEGX at pH 5.6 is approximately 35.34 ％. And the calculated α-helicity atpH 1.6, 9.7, and 10.6 are significantly lower than that of the protein at pH5.6. These data indicate at pH 5.6 the molecule is most compact and partialα-helicity structure will lose following the changes of pH. At pH 10.6 theα-helicity structure lost 72.3 ％, which suggests that the molecule is muchmore incompact and unstable at the alkaline pH. The thermal stability of EGX at various pH values is much different.The EGX remains about 80 percent activity in the pH range of 5.6 to 7.5 and 63吉林大学硕士学位论文 李文颖drops only on either side of this pH range. And at pH 2.7, 3.5, 9.5, 10.5, 11.1and 11.9 all the activities of the enzyme were lost. The data indicates thatthe structure changes of EGX are much more significant out the pH range of5.6 to 7.5. The molecule becomes less compact and more hydrophobic,...