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

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.

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High-Level Gene Expression for Recombinant Penicillin Acylase Production Using the araB Promoter System in Escherichia coli

Biotechnology Progress ◽

10.1021/bp060135u ◽

2006 ◽

Vol 22 (6) ◽

pp. 1518-1523 ◽

Cited By ~ 3

Author(s):

N. Narayanan ◽

Y. Xu ◽

C.P. Chou

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Penicillin Acylase ◽

High Level ◽

Promoter System

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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

Zeitschrift für Naturforschung C ◽

10.5560/znc.2014-0061 ◽

2014 ◽

Vol 69 (11-12) ◽

pp. 479-487 ◽

Cited By ~ 2

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.

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