| Summary: | 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.
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