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Isolation and Indentification of a Thermophilic Cellulolytic Anaerobic Bacteria Strain B2 and Its Molecular Biological Studying

Author: HuGuoQuan
Tutor: ZhangChaoWu
School: Sichuan University
Course: Nutrition and Food Hygiene
Keywords: thermophilic anaerobic bacteria cellulolytic bacterium 16S rDNA phylogenetic analysis endoglucanase gene(EDgene) cloning Hungate’ s anaerobic techniques
CLC: Q939.99
Type: PhD thesis
Year: 2004
Downloads: 830
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Abstract


[Objective] 1. Through selective enrichment culture, several thermophlic cellulolytic anaerobic bacterium were isolated from hot spring and oil well.2.Study on biological respectives of the isolated strains, such as medium resources, the main fermentation products and enzyme chracterstics for cellulose.3. Studied these strains prospects of utilize through fermentation these isolated strains on the cellulose , such as distiller’ s grains and rice straw, and effect peel oil off .4. The isolated strains were identified by their physiological and morphological characteristics, molecular methods were used to identified the species, molecular methods include the use of 16S rDNA and PCR amplayfided .5. Genomic library of the best isolated strain was constructed. DNA extraction → partially digestion by restriction enzymeEcoR I → ligation with plasmid pUC18 → transformation E. coli JM1096. Endoglucanase gene cloned from Genomic library. Recombinants screening by the technique of Congo Red plates.[Methods] 1. Isolation and screening methods for thermophilic cellulolytic anaerobic bacteria2. Media can be added as solutions to individual Hungate-tube, serum bottles and 5L fermentation vessels, during addition media,Hungate’ s anaerobic techniques can be used. 3.roll tubes technique.4. The isolated strains were identified by their physiological and morphological characteristics, molecular methods were used to identified the species, molecular methods include the use of 16S r DNA and PCR amplayfided .5. Techniques of Assay the active of cellulases, such as CMCase, Ci > Cx and £ -glucosidase.6. Studied these strains prospects of utilize through fermentation it on the cellulose , such as distiller’ s grains and rice straw, and effect peel oil off .7. Cloning endoglucanase gene used by the technique of Congo Red plates.8. The expression recombinant with foreign DNA fragments protein was analyzed by SDS-PAGE .[Results] 1 Isolation of thermophilic cellulolytic anaerobic bacterium and studing their utilize prospects. 1. 1 Several thermophlic cellulolytic anaerobic bacterium were isolated from hot spring and oil well. B2, B6, B7 have better ability in degrading cellulose among these cultures. They are all strictly anaerobic bacterium, and cells are rods, motion in PY media. 1.2 The growth temperature of B2 , B6 and B7 occurre range 40~ 80°C, and an optimal is 60~65°C, 65°C and 60~65°C respectively. The optimal pH are 7.0,8.0 and 8.0 respectively. Their index stage of growth are 34~36h, 28h and 8~12h respectively. 1. 3 The results for carbon source showed that star and suger arebetter than the others, and the enzyne activitis of B2 and B6 in glucose is bigger than the filter paper.1. 4 The temperature range of activity for B2 , B6 and B7 cellulase is 50~110°C. There are cell-in and cell-out enzyme in the cellulase system of B2 , B6 and B7 .1.5 Through the experiments of utilize for B2 , B6 and B7 , it is showed that,1) Cells and metabolize products can emulsify crude oil.2) Their growth and metabolize can peel crude oil off.3)They can ferment the cellulose and produce the organic acid , such as actate, acrytic acid and butyric acid. 2 Indentification and phylogenetic analysis of a thermophilic cellulolytic anaerobic bacteria strain B2 and its enzyme activity characteristic.2.1 The thermophlic cellulolytic anaerobic bacteria strain B2 was isolated from hot spring in Yunnan, China. Cells are straight rods 0. 4 u m by 2um~4um, occurring both singly and in pairs. Gram staining reaction was negative. Endospores were never observed. Growth strictly anaerobic, occurring over the temperature range 40~80°C with an optimum of 65°C.The pH range was 4~8, the optimum pH being 7.0. On cellulose agar colonies were 2~4mm in diameter, and with cream colour. Monosaccharides, disacchrides and polysaccharides serve as fermentable substrates. The main fermentation end products on eel lose medium are actate and ethanol are also detected.2. 2 The results show that the ratio among Ci> Cx and {3 -glucosidaseis 1:9:10,the optimum temperature are 80°C> 80°C and 70 °C, respectively. Meanwhile,. Cx possesses high hot-stability.2. 3 The phylogenetic analysis based on 16S rDNA suggested strain B2 was the closest relative of Therwoanaerobacter ethanalicus with 99.8% sequence similarity. The strain B2is the species Thermoanaerobac ter.3 Construction of B2 genomic library and cloning of endoglucanase gene fragments.3. 1 Genomic DNA of B2 was extracted and was partially digested by restriction enzyme EcoRI, and ligated with plasmid pUC18 by T4DNA ligase. After transformation F. coli JM109, the genomic library was constructed and 6. 3X103 recombinants was obtained. The ratio of the recombinants which formed transparent zones on the Congo Red plates was 23. 5%. The results after digested by restriction enzyme EcoRI revealed that the recombinants which had been picked out contained foreign DNA fragments. The results showed that the endoglucanase gene has been cloned.3.2 No plasmid was detected in strains B2.3.3 Under heat induced(37°C), the expression recombinant protein was analyzed by SDS-PAGE and its relative molecular mass is 46kD. Our results may lay a foundation for the further exploitation and utilization of cellulose.[Conclusion] 1. Three thermophlic cellulolytic anaerobic bacterium were isolated from hot spring and oil well. Their optimal growth temperature occurre range 60~65°C. 2. Through the utilizing experiments , it is showed that,they can emulsify crude oiland and peel crude oil off.3. The thermophlic cellulolytic bacteria strain B2 Growth strictly anaerobic, occurring over the temperature range 40—80°C with an optimum of 65°C. the ratio among d> Cx and P-glucosidase is 1:9:10, the optimum temperature are 80°C> 80°C and 70°C,respectively. Meanwhile, Cx possesses high hot-stability.4. No plasmid was detected in strains B2.5. the genomic library was constructed and 6. 3X1O3 recombinants was obtained.6. Endoglucanase gene cloned from Genomic library.

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