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Bioprocess optimization using metabolic engineering approach

หน่วยงาน สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์

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ชื่อเรื่อง : Bioprocess optimization using metabolic engineering approach
นักวิจัย : Wanwisa Skolpap
คำค้น : Chemical engineering , Bioprocess , Metabolism , Amylases-alpha , Rhamnose , Threonine , Isoleucine
หน่วยงาน : สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์
ผู้ร่วมงาน : -
ปีพิมพ์ : 2546
อ้างอิง : Ph.D., University of Waterloo (Canada), 2003, 313 pages , 0612830233 , http://dspace.library.tu.ac.th/handle/3517/4076 , http://dspace.library.tu.ac.th/handle/3517/4076
ที่มา : -
ความเชี่ยวชาญ : -
ความสัมพันธ์ : -
ขอบเขตของเนื้อหา : -
บทคัดย่อ/คำอธิบาย :

The metabolism of the recombinant Bacillus subtilis ATCC 6051a, producing extracellular α-amylases as desirable products and proteases as undesirable products, was studied as a model system to understand metabolic regulation and product distribution. The objective was to optimize the specific productivity of a recombinant enzyme by applying a metabolic engineering approach. Measured extracellular metabolites included dry cell mass, α-amylase and protease activity, and fermentation by-products. Shake-flask, batch, and fed-batch cultures were carried out to develop a stoichiometry based metabolic flux model. The defined medium containing starch and citrate as carbon sources was chosen as a baseline due to a 2.8-fold improvement of maximum α-amylase activity over a Luria broth (LB) complex medium in shake-flask studies. The specific rate of dual enzyme formation was mainly non-growth associated. The metabolic flux model was constructed to provide detail on the amino acid and bioenergetic fluxes. Both the results of experiments and flux estimates indicated that system optimization should be carried out on the basis of nitrogen limitation, the variation of C:N ratios and that the addition of key amino acids should have a significant effect on the synthesis and distribution of both enzymes. Rhamnose was not a good α-amylase inducer. The most favorable C:N ratio for α-amylase and protease synthesis was 8-9.5 and 12-12.5, respectively. In the presence of citrate, flux partitioning via pyruvate to oxaloacetate, an anapleoric reaction, was initiated to enhance the synthesis of amino acids derived from oxaloacetate. Under oxygen limitation ethanol and organic byproducts such as lactate, acetate, and formate were produced and had an influence on ATP generation. The metabolic flux estimation showed that the flux leading to the synthesis of the amino acids threonine and isoleucine was particularly attenuated. It was found that the addition of threonine and isoleucine in batch culture could enhance α-amylase synthesis more than 2.5-fold. Moreover, in fed-batch culture, α-amylase production was enhanced over 2.6-fold while protease production showed no significant change if threonine and isoleucine were fed continuously. A mixture of proline and isoleucine were not protease inhibitors, but it delayed the onset of protease synthesis and enhanced cell growth rather than α-amylase synthesis. During the feeding period with the increase of α-amylase synthesis the amino acid flux ratios with respect to their corresponding precursors and the fluxes associated with energy generation were relatively constant while the specific growth rate was maintained at low value. (Abstract shortened by UMI.)

บรรณานุกรม :
Wanwisa Skolpap . (2546). Bioprocess optimization using metabolic engineering approach.
    กรุงเทพมหานคร : สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์ .
Wanwisa Skolpap . 2546. "Bioprocess optimization using metabolic engineering approach".
    กรุงเทพมหานคร : สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์ .
Wanwisa Skolpap . "Bioprocess optimization using metabolic engineering approach."
    กรุงเทพมหานคร : สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์ , 2546. Print.
Wanwisa Skolpap . Bioprocess optimization using metabolic engineering approach. กรุงเทพมหานคร : สถาบันวิจัยและให้คำปรึกษาแห่ง มหาวิทยาลัยธรรมศาสตร์ ; 2546.