| Along with the population explosion, growth of living standard and acceleration of the process of industrialization, global energy demand increases rapidly. Energy security, oil consumpt ion, global warming and the other problems prompt international research on alternative energy sources. Development of cellulosic ethanol has become study focus. Natural lignocellulose, which bases on cellulose, hemicellulose and lignin, is accepted the most abundant and cheapest biomass resource. Cellulose biodegradation relies on cellulase that produced by microorganism. Srains transform by mutagenesis and genetic engineering could rise the enzyme activities, which is a effective measure for efficient producing strain selection.This study aims at finding a wild type fungi who has remarkable cellulase production capacity. Microorganism comes from rotten weed, leaf and straw. Take natural cellulose as sole carbon source for enrichment culture. Steaking and switching in liquid culture medium for screen high-cellulase-activites strain according to FPA. Identify the strain using 26S rDNA D1/D2 region sequencing. A efficient cellulase producing strain identified belonging to Trichoderma sp is named as Trichoderma sp.A6. Optimize the culture system to improve the cellulase activities. Adding lactose of 10g/L arise the FPA to 0.56IU from 0.16IU. The FPA reach the peak in the entire cultivation course at the 2nd day. Lactobionic acid is useless for FPA enhance. Tween80 can help weakly for 4th day enzyme activities. Yeast extract of 15g/L arise the 2nd day FPA to 0.92IU from 0.60IU, well yeast-extract-Tween80-union-adding works no better than their sole adding. Finally we confirm the optimum condition of culture as lactose of 1 Og/L and yeast extract of 15g/L adding togetherUV mutate the strain Trichoderma sp.A6 and make plenty of screen to get high-cellulase-prodution strain 2-3D. Purify it and insure its hereditary stability. Fetch crude enzyme after shake-cultivation and liquid-submerged-fermentation and to assay the enzyme activities. CMCase reaches 1.44IU and FPA 1.04IU. Suction filtrate the crude enzyme, cement out the zymoprotein by tannin, vacuum-lyophilize it to get enzyme powder whose activities arises a lot, which is 5 times to origin. Strain 2-3D has a short incubation period, possesses high cellulase activities and shows no notable undulate of enzyme activities after repeatable subculture.Make high-throughput-sequencing towards the strains to get their transcriptome. Average length of all sequences is 98bp.15763 unigenes whose total size reaches 20.37Mb, are jointed in de novo way. Gross of transcripts is 21594. Their average length is 1572.65bp. GC-content appears gaussion distribution from 50% to 60%.73.53% of all genes are annotated to Trichoderma Reesei QM6a.109 genes are annotated to’Amino acid biosynthesis metabolism’. Roles of gene ko00010 and gene ko00190 through metabolism are described. Compared with origin strain expression,1549 genes reduce,267 genes up-regulate,1816 genes change in total. DNA replication and metabolism, xylan anabolism and catabolism, hemicellulase metabolism all shows differential expression. DNA replication pathway and yeast cell cycle pathway express extremely remarkable... |
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