| With the development of economiand social progress, the fossil fuels are gradually exhausting,and seeking more secure, environmentally friendly and cost-effective new renewable energy to replace fossil fuels as part of the current problems is to besolved. Cellulose is the most abundant renewable resource in the world. Cellulose can be transformed to the fermentable syrupagriculture composed polysaccharides and monosaccharides which can be fermented to the clean liquid biofuels alternative to fossil fuels. However, the high cost of cellulase production is the bottleneck of conversing cellulose to ethanol.Thereore, seeking cellulase high strain and understanding the mechanism of activity improvement has important economic and social significance.In this study, more than 14 thermophilic bacteria were successfully isolated from Yongtai hot spring of Fujian province by congo red staining and determining the enzyme activity. And two of them were identified by 18S rDNA sequencing analysis. YT-40-1 was belong to Candida parapsilosis.And WJM-4-G was belong to Trichosporon asahii. Fermentation conditions of two superior cellulose decomposing strains YT-40-1and WJM-4-G were further studied. The result shows when tempreture and pH reached the 50℃and 4, culture time was 72 hours, the total celluase activity of YT-40-1 reched its peak. When the culture time was 24h, it began to go to the logarithmic growth phase. After cultured 24h, WJM-4-G went into the logarithmic growth phase, and the optimum fermentation conditions follow. The temperature is 40℃, the culture time is 60h and the optimum initial medUm pH is 4 or 7.Physical and chemical mutagenesis were done on strains YT-40-1, WJM-4-G and CC-1. We got two mutants, 1-0.3-1 and 1-0.3-4, which had higher cellulase activity, from the srain YT -40-1 in LiCl mutagenesis. Theβ-glucosidase activity of strain 1-0.3-1 was 669.0U, and its CMCase activity was 121.9U. The activity increased by 96.1% and 72.1% than the original strain. Theβ-glucosidase activity and CMCase activity of 1-0.3-4 were respectively 717.5 and 125.3, higher than the original strain by 110.3% and 74.0%. When the gas flow rate was 15L/min and the mutation time was 7min, we got another mutant, YT-40-1(15-7-Ⅲ), from YT-40-1 in plasma mutagenesis. Theβ-glucosidase activity of strain YT-40-1(15-7-Ⅲ) was 571.2U higher than the original strain by 27.2%, and its CMCase activity was 152.8U increasing by 73.0% than the original strain. We got a mutant when we treated the strain WJM-4-G in DES for 20min. Itsβ-glucosidase activity 555.9U, and the CMCase activity reached 148.7U. When we treated the strain WJM-4-G in DES for 25min, we got a superior cellulose decomposing mutant, and theβ-glucosidase activity and CMCase activity were 550.2U and 144.1U respectively. After the strain WJM-4-G was put in ultra-violet ray for 10min, and then cultured on medUm which had LiCl, we got another mutant G-10-Li. Theβ-glucosidase activity of strain G-10-Li was 438.1U which was higher 27% than the original strain, and its CMCase activity was 184.0U which increased by 131.4%. In ultra-violet ray mutagenesis for 10min we got a mutant from stain CC-1. We named the mutant CC-1-10. Theβ-glucosidase activity and CMCase activity of strain CC-1-10 were 511.1U and 155.6U respectively which increased by 20.4% and 55.6% respectively.Whole cell protein SDS-PAGE analysis and random amplified polymorphic DNA(RAPD)showed that the original strain YT-40-1 and the mutant YT-40-1(15-7-Ⅲ) had significantly different protein, DNA bands were also different.It exactly demonstrated at protein level and genomic level that YT-40-1(15-7-Ⅲ) was surely a mutant of YT-40-1. |
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