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Studies on Amino Acid Replacement and Inhibitory Activity of a β-Lactamase Inhibitory Peptide

Liping Xie, Mingfei Xu, Tao Yang, Chunbao Zhu, Baoquan Zhu, and Youjia Hu*

State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 Zhongshan Road (North No. 1), Shanghai 200437, China; fax: 86-21-3505-2484; E-mail: bebydou@hotmail.com

* To whom correspondence should be addressed.

Received August 31, 2009; Revision received September 28, 2009
An SHV β-lactamase gene was amplified from a β-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity of both TEM-1 and SHV β-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1-C, P1-G, and P1-R, respectively. The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV β-lactamase were determined. All these β-lactamase inhibitory peptides could inhibit the activity of both β-lactamases, while the mutant peptides showed stronger inhibitory activities against TEM-1 β-lactamase than against SHV β-lactamase. Inhibition data suggested that P1-A improved the β-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria–Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study gives us a good candidate for development of novel β-lactamase inhibitors.
KEY WORDS: β-lactamase inhibitory peptide, β-lactamase, amino acid replacement, Klebsiella pneumoniae, inhibitory constant

DOI: 10.1134/S0006297910030107