Characterization of the T7 promoter system for expressing penicillin acylase in Escherichia coli

2006 ◽  
Vol 72 (3) ◽  
pp. 529-536 ◽  
Author(s):  
Yali Xu ◽  
Stefan Rosenkranz ◽  
Chiao-Ling Weng ◽  
Jeno M. Scharer ◽  
Murray Moo-Young ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Penicillin Acylase ◽  
T7 Promoter ◽  
Promoter System

Characterization of the regulatory region of the Escherichia coli penicillin acylase structural gene

Gene ◽  
1986 ◽  
Vol 50 (1-3) ◽  
pp. 119-122 ◽  
Author(s):  
F. Valle ◽  
G. Gosset ◽  
B. Tenorio ◽  
G. Oliver ◽  
F. Bolivar
Keyword(s):  
Escherichia Coli ◽  
Structural Gene ◽  
Regulatory Region ◽  
Penicillin Acylase

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

Expression and Characterization of Two β-Galactosidases from Klebsiella pneumoniae 285 in Escherichia coli and their Application in the Enzymatic Synthesis of Lactulose and 1-Lactulose

2014 ◽  
Vol 69 (11-12) ◽  
pp. 479-487 ◽  
Author(s):  
He Wang ◽  
Ruijin Yang ◽  
Xiaoyan Jiang ◽  
Xiao Hua ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Enzymatic Synthesis ◽  
Ph Value ◽  
Optimum Temperature ◽  
Recombinant Enzymes ◽  
T7 Promoter ◽  
Expression In Escherichia Coli ◽  
Higher Temperature

Abstract The two genes lacZ1 and lacZ2 from Klebsiella pneumoniae 285, encoding β-galactosidase isoenzymes II and III (KpBGase-II and -III), were each cloned downstream of a T7 promoter for expression in Escherichia coli BL21(DE3), and the resulting recombinant enzymes were characterized in detail. The optimum temperature and pH value of KpBGase-II were 40 °C and 7.5, and those of KpBGase-III were 50 °C and 8.0, respectively. KpBGase-III was more stable than KpBGase-II at higher temperature (>60°C). Both β-galactosidases were more active towards o-nitrophenyl-β- D-galactopyranoside as compared to lactose. The enzymatic synthesis of lactulose and 1-lactulose catalyzed by KpBGase-II and KpBGase-III was investigated. Using 400 g/L lactose and 200 g/L fructose as substrates, the resulting lactulose and 1-lactulose yields with KpBGase-II were 6.2 and 42.3 g/L, while those with KpBGase-III were 5.1 and 23.8 g/L, respectively. KpBGase-II has a potential for the production of 1-lactulose from lactose and fructose. Like other β-galactosidases, the two isozymes catalyze the transgalactosylation in the presence of fructose establishing the β-(1→1) linkage.

scholarly journals Characterization of the β-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

2000 ◽  
Vol 13 (12) ◽  
pp. 857-863 ◽  
Author(s):  
Wynand B.L. Alkema ◽  
Charles M.H. Hensgens ◽  
Els H. Kroezinga ◽  
Erik de Vries ◽  
René Floris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Penicillin Acylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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