| Cellulose is the most abundant renewable resource in the world. Cellulase is a kind of biological catalyst that can completely degradate cellulose to glucose enzymatically through the synergistic action of several types of enzymes. Cellulase is widely used in bioenergy, food, textile, feeds and so on. Many varieties of inserts and plant can produce cellulase, but industrial celluase is almost secreted by microorganisms. Producing cellulase by fungi has many adventages, including simple cultivation, full enzymes, high yield, easy extraction. Furthermore, the cellulase from thermophilic fungi has a better application prospects because of its good heat resistance. The cellulase production fungi directly isolated and purified from nature present low enzyme activity and little enzyme production. Therefore, using mutagenesis and molecular biology techniques to modify fungi is necessary to further breed high cellulase yield thermophilic fungi. As the purpose of this paper, the main results of the experiment are as follow:(1) Three produce cellulase thermophilic fungi named M40-1, M40-2, and M40-3 were successfully isolated and purified from Yongtai hot spring of Fujian province by Congo red staining and determining the enzyme activity. Three stains were identified by ITS rDNA sequencing analysis and belong to Aspergillus fumigates. The highest cellulase yield stains was M40-1. When it got optimum fermentation conditions:the culture time was 48 hours, the temperature was 40?, and the pH was 5.0, we will get the highest CMC activity and ?-glucosidas activity, which were 5.64 U/ml and 4.31U/ml.(2) We established the protoplast preparation, protoplasts regeneration and protoplasts fusion of produce cellulase thermophilic fungi YT-40(Candida parapsilosis) and its positive mutagenesis strain:protoplasts were prepared from the mycelia harvested after 24 hours culture in rich culture medium. Protoplasts were isolated by using an enzyme combinations composed of 3mg/ml Snailase and 3mg/ml cellulase for 1 hour. The final population of protoplasts in total cells was 75%, and a high regeneration ratio of 80% was occurred on the optimal regeneration medium.Then the positive mutagenesis strains of YT-40 were prepared and fused in system composed of 35?,35% PEG-6000,0.01M CaCl2.(3) YT-40 and its protoplast were mutated by plasma, UV & LiCl, DES and compound mutagen. We got eight positive mutants from single mutagen, whose CMC enzyme activity increased 76.56% to 122.45% than YT-40. The CMC enzyme activity of two positive mutants from compound mutagen increased 156.67%(normal state) and 150.64%(state of protoplas). The eight positive mutants from single mutagen were used as parents for genome shuffling. After screening on plates and determining the enzyme activity, one fusant named F14 with significant improvement were acquired. It could produce cellulase as much as 176.74% of the original strain YT-40. Besides, through the five times of pass-generation enzyme production nurturing experiments, they can prove that they have stable cellulose activity as well as high genetic stability.(4) The optimum fermentation conditions to produce CMC enzyme of YT-40: the culture time was 24 hours, the temperature was 40?,and the pH was 6.0. Under these conditions, the CMC activity can reach the top, which was 6.27 U/ml. The optimum fermentation conditions to produce CMC enzyme of F14:the culture time was 24 hours, the temperature was 45?, and the pH was 5.0. Under these conditions, the CMC activity can reach the top, which was 16.70 U/ml.(5) Analysing the comparisons of molecular characteristics between the fusant F14 and the original strain YT-40 by SDS-PAGE and RAPD. |
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