Construction of γ-PGA-producing strain bychromosomal integration in Bacillus subtilis andoptimization of fermentative production

• Main Authors: Jun-Ming Zheng, 鄭浚鳴
• Other Authors: 葉娟美
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/w94ut2

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 102 === Poly-γ-glutamic acid (γ-PGA) is a versatile high molecular biopolymer material, which has been applied in food, medical, cosmetic, animal feed,and wastewater industry. Enhanced production of γ-PGA is highly recommended. In order to enhance the screening of high γ-PGA yielding strain and quantitation of γ-PGA. A rapid quantification of γ-PGA was established in the beginning of this study. CTAB binds specifically to γ-PGA and form a water-insoluble, highly dispersed micelle-like complex, resulting in an increase in turbidity. The turbidity-based calibration curve of γ-PGA was established as y = 0.0055x – 0.0349 (x and y represent the concentration of γ-PGA and the mixtures turbidity at 400 nm) with a good linearity. The turbidimetric method has advantages of convenience , simplicity and good repeatability and can be used for γ-PGA concentration detecting in the fermentation broth. The host Bacillus subtilis WB800, which possesses the γ-PGA synthesizing genes, pgsBCAE, on its chromosome cannot produce γ-PGA. The efficient constitutive or inducible synthetic expression control sequence (SECS) was introduced into the upstream region of the pgsBCAE genes, resulted in γ-PGA-producing B. subtilis transformants. The transformant strain B. subtilis Dc8006 stably produced high levels of γ-PGA in medium A without extra glutamate supplement. To evaluate the effect of different culture parameters on production of γ-PGA, Plackett–Burman factorial design was preceded. Twelve varients were examined for their significance on γ-PGA production. Based on statistical pre-optimized medium analysis, optimized medium PBD were subjected for fermentation, and achieved 35.2 g/l γ-PGA yield, which is 1.5 times than the original medium A.