Enhancing Cellulase Production By Mutagenic Breeding, And Analysis Of The Mechanism Of Differences In Cellulase Production Of Trichoderma Longibrachiatum

Second generation bioethanol produced from various lignocellulosic materials is one of the most valuable renewable energies. However, the activity of most cellulases is relatively low. Also the supply of cellulase has failed to keep pace with demand. In order to get strain mutants that have higher celluase production, this research carried out the mutation breeding of Trichoderma longibrachiatum A002 using the UV light and microwave. Further, the phenotype change of T. longibrachiatum mutant that produces cellulase, was analyzed with the proteomics techniques. The main results were as follows:(1) Cellulase-producing fungus T. longibrachiatum A002 was utilized as material for mutagenesis by microwave and ultraviolet to enhance the cellulase production. Congo red medium was used to screen the strains that had stronger ability to produce enzymes. The characteristics of carboxymethyl cellulose (CMCase) under the condition of liquid fermentation was evaluated, and the optimum culture time, rotary shaker and temperature for CMCase activity of T. longibrachiatum were 96h, 180 r/min and 30℃, respectively. After the compound mutagenesis by microwave and ultraviolet, ten mutant strains (M01-M10) were selected and their CMCase activities were assayed. Six of them (M01, M05, M06, M08, M09 and M10) had significantly stronger ability to produce enzymes than the normal wild type, but only four of them (M01, M05, M06 and M08) were very stable for a long period up to 9 generations to produce cellulase. Among them, the enzymatic activity of M06 was the highest, which was 1.66 times of the original strain. Based on the optimized treatment in the cellulase production of the mutant srtains M06 on solid-state fermentation conditions, the results showed that the initial pH was 3, the Ratio of material to water was 5, and optimum temperature and culture time were 34℃ and 96 h。(2) On purpose of expounding the phenotype change of T. longibrachiatum mutant that produces cellulase. Protein profiles of T. longibrachiatum A002 and its mutant M06 were detected by 2-DE.58 unique spots were identified by LC-MS/MS, 18 of them were predict proteins or hypothetical proteins, predicted by the database of genome. Most of the protein functions were connected with carbohydrate and protein metabolism, substance transportation and signal transduction of Trichoderma. Among these proteins, the expression of cellulase was enhanced, which was the most important enzymes in process of lignocellulosic degradation.Some of proteins were involved in two G-protein-mediated signal transduction pathway, G protein-coupled phosphoinositide pathway and G protein Ras-mediated MAPK pathway. These two signal transduction pathways involved in the induction of gene expression and regulation of cell division.In summary,this study, cellulase-producing fungus T. longibrachiatum A002 was utilized as material for mutagenesis by microwave and ultraviolet, screening a cellulase producing ability and stable genetic mutants M06.Through differential proteomics made a preliminary analysis of the enhanced mechanism of mutants M06 cellulase production. Infer that there were two kinds of G-protein-mediated signaling pathways, G-protein-coupled phosphoinositide pathway and the MAPK pathway of G-protein Ras-mediated protein. Some of the proteins expressions were changed, result in enhanced expression of cell secretion and inhibited cell growth and its division.