Studies On Cellulase Induced By Waste Molasses Of Ethanol From Trichoderma Knoningii

The waste molasses of ethanol, deep brow, high acid, condensed waste organic water, is one of the main puollution sourses from sugar refineries. During the comprehensive disposal of the waste performed by Food and Fermentation engineering Institute of Guangxi University, the waste molasses of ethanol was found to be helpful to the excretion of cellulase from Trichoderma Knoningii. The further studies on cellulase from Trichoderma Knoningii affected by the waste are achieved in this thesis.Firstly, the enzymic characteristics of cellulase from waste molasses medium, including FP, EG, BG and CBH are determined in this thesis. The optimum pH for catalyzed reactions of FP, EG, BG and CBH are 4.8, 4.8, 4.8, 5.0, respectively. The optimum temperature for catalyzed reactions of FP, EG, BG and CBH are 50℃, 70℃, 60℃, 55℃, respectively. The michaelis constant of EG, BG and CBH, as well as the effects of substrates, dilutions and ions on FP, EG, BG and CBH are assayed.Secondly, purification and isolation of cellulase are performed in the thesis. By means of ammonium sulphate precipitation, anion exchange chromatography column DEAE cellulose 52 and DEAE sepharose CL-6B, the recoveries of EG and BG reach 2.06% and 65.11%, purity of EG and BG reach 24.55 times and 169.84 times, specfic activities of EG and BG reach189.48IU per mg protein and 656.28IU per mg protein, respectively.Thirdly, the mechanism of cellulase induced by waste molasses of ethanol from Trichoderma Knoningii is investigated. It's the function of inducement, not function of nourishment that cellulase is excreted excessively from Trichoderma Knoningii on the medium of waste molasses. The activity of FP is 4.8 times as much as that of Mandels medium, comparative with EG, BG and CBH are 3 to 5 times as much as that of Mandels medium. Analyzed by SDS-PAGE, waste molasses of ethanol can induce two proteins over-Expression from Trichoderma Knoningi. These proteins, named P17.8andP72.9 according to molecular weight 17.8KD, 72.9KD respectively, have no activities of EG, BG and CBH. So P17.8and P72.9 have not activity of cellulase. They are not cellulase.