Secretion and production of alpha‐amino‐acid esterase using aae gene clone in Escherichia coli.

• Main Authors: Chen, Nien-Xsuan, 陳年軒
• Other Authors: Lin, Wen-Po
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/59596260297825895004

碩士 === 國防醫學院 === 微生物及免疫學研究所 === 99 === X    English Abstract Background & Aims: The α–amino-acid esterase (AAEase) performing alpha-aminoacyl coupling to a number of amine nucleophiles that can catalyze synthesis of β-lactam antibiotics has been described in many bacteria such as Acetobacter turbidans, Acetobacter pasteurianus, Xanthomonas citri, and Gluconobacter oxydans.The recombinant plasmid pLC802 bearing aae gene cloned from G. oxydans 621H was transformed in E. coli BL21(DE3) host cells harboring the T7 RNA polymerase gene under lac UV5 promoter control; afterward, the aae gene overexpressed by the isopropyl-β-D-Thiogalactoside (IPTG) in E. coli substituted for D-phenylglycine as an inducer originated in parent strain. According to above, we want to study the cellular secretory mechanism of AAEase in E. coli and the optimal fermentation process for production yield. Methods & Results: Western blotting and bioautographic result revealed that the new gene clone, E. coli BL21 / pLC 802, expressed and secreted activity AAEase . Next, we separated the protein fraction of cytoplasm and periplasm by osmotic shock method. Most of the active form located in periplasm, whereas premature form aggregated in cytoplasm. The fermented process on cell-growing culture (TB medium, pH 7.0, 200 rpm shaking speed, and growing  temperature at 37℃) and gene-expressed culture (0.33 M IPTG, 150 rpm shaking speed, fresh culture medium replacement before IPTG induction, and inducing temperature at 28℃ for 8 hours) was optimal condition for AAEase production. Conclusion: According to above, the results revealed the AAEase is a sec-targeting protein though the post-translational processing mechanism to periplasm and maturation. Using the optimal fermentation process, the AAEase production yield of the gene clone is 300 times more than parent strain.