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The defense date of the thesis is 2012-09-04
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URN etd-0903112-143216 Statistics This thesis had been viewed 1340 times. Download 0 times. Author Yu-fei Chan Author's Email Address No Public. Department Bioengineering Year 2011 Semester 2 Degree Master Type of Document Master's Thesis Language zh-TW.Big5 Chinese Page Count 77 Title Investigation of Lactose-Induced Production of β-glucosidase from Recombinant Escherichia coli Keyword recombinant Escherichia coli β-Glucosidase Lactose Lactose β-Glucosidase recombinant Escherichia coli Abstract β-Glucosidase is a cellulolytic enzyme. Cellulose is composed of glucose units by β-1.4 glucosidation with a combination of linear polymer chains. Enzymatic decomposition of cellulose is initialized with endo-cellulase (endo-β-1 ,4-glucanase) which cuts cellulose chains into shorter units. Cellulohydrase hydrolyzes cellulose into small glucosidic molecules with reducing ends, such as cellobiose. In the final step, β-glucosidase cuts cellobiose into glucose molecules. Generally microorganisms can not directly get energy by consuming cellobiose.
The present work is engaged in the production of β-glucosidase by fed-batch fermentation of a recombinant E.coli. β-Glucosidase encoded gene from a thermophilic anaerobe was constructed in pET21-a plasmid. Not only isopropyl-β-D-thiogalactoside (IPTG) but also Lactose were used for the gene expression. Preculture was carried out using a 5-L fermenter with a working volume of 3 L, at 25℃, 300 rpm, 1 vvm and pH being controlled in a range of 7.1-7.4. The induction of β-glucosidase was carried out by transferring 100 mL of preculture into a 250-mL flask aseptically. After addition of different inducer, the flask was incubated in a 25℃shaker at 150 rpm for 64 h.
The fermentation was carried out by varying the composition of the culture medium composition, the concentration of inducer, the preculture time and induction time. When Luria Bertani (LB) was used as the culture medium, a maximum β-glucosidase activity at 17.89 U/mL was obtained after a 6-h preculture and a 64-h induction by adding 8 mM lactose as inducer.
When Terrific Broth (TB) was used as the culture medium, the highest β-glucosidase activity up to 82.04 U/mL was obtained after a 8-h preculture and a 64-h induction by adding 8 mM lactose as inducer. β-Glucosidase activity obtained using phosphate-reinforced TB as culture medium was 4.59-fold higher than that LB was used.
Advisor Committee Dey-Chyi Sheu - advisor
Chi-Tsai Lin - co-chair
Wang, Chung-Yih - co-chair
Files Date of Defense 2012-07-24 Date of Submission 2012-09-04