Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/2935
Title: Properties of novel beta-lactamase enzymes
Authors: Cheung, Wai-ling
Keywords: Beta lactamases
Hong Kong Polytechnic University -- Dissertations
Issue Date: 2000
Publisher: The Hong Kong Polytechnic University
Abstract: Beta-lactamases are enzymes produced by pathogenic bacteria to destroy beta-lactam antibiotics, such as penicillins and cephalosporins. Most cases of antibiotic resistance are caused by the presence of these secretory enzymes. Despite many years of intensive studies, amino acid residues involved in this important hydrolytic action have not been sufficiently defined. Class A beta-lactamases are most commonly employed by bacteria to hydrolyze various beta-lactams. Although they are similar to each other at levels of primary protein sequence and tertiary structure, they behave diversely with respect to substrate specificity and activity. In order to identify the regions of the polypeptide chain containing amino acid residues that determine the specific behavior of class A enzymes, novel hybrid beta-lactamase genes were previously created by Leung et al. (Protein Eng. 6, pp.66 Suppl.) using in vivo intramolecular homologous recombination. These hybrid genes have their N-terminal and C-terminal coding moieties derived respectively from the beta-lactamase I (PenPC) gene of Bacillus cereus and the Bacillus licheniformis beta-lactamase (PenP) gene. In this present study, five of these genes were selected for detailed analysis. The enzymes were over-expressed in an efficient Bacillus subtilis expression and secretion system and purified from the culture supernatant. The five hybrid beta-lactamase enzymes studied in this project are named as Hyb 8(C²⁸³L¹²), Hyb 7 (C²⁴⁶L⁴⁹), Hyb 5 (C²¹²L⁸³), Hyb 12 (C²⁰⁰L⁹⁵) and Hyb 6 (C³⁷L²⁵⁸). For example, C³⁷L²⁵⁸ indicates that out of the 295 amino acids, the first 37 are derived from beta-lactamase I of B. cereus, the next 258 (residue 38-295) from the beta-lactamase of B. licheniformis.
According to the enzyme activity studies using penicillin G and penicillin V as substrates, the seven enzymes can be divided into two groups: Group 1 contains the wild-type beta-lactamase I (PenPC), Hyb 8, Hyb 7, Hyb 5 and Hyb 12; Group 2 includes the wild-type B. licheniformis enzyme (PenP) and Hyb 6. Group 1 members can hydrolyze these penicillin type substrates much more efficiently than Group 2. However, when cephaloridine, a cephalosporin type substrate, was used as the substrate, Group 2 enzymes were much more active than those in Group 1. Hyb 6 (C³⁷L²⁵⁸) contains the first 37 amino acid residues of beta-lactamase I and the last 258 residues of the B. licheniformis enzyme. The incorporation of these 258 residues in the C-terminus caused the hybrid enzyme to behave in the same way as the B. licheniformis enzyme in terms of profiles of substrate specificity, thermosensitivity, pH-activity, but not thermostability. However, proteins Hyb 8, Hyb 7, Hyb 5 and Hyb 12 all showed similar kinetic properties to the wild-type beta-lactamase I. This suggests that amino acid residues 201-291 of beta-lactamase I can be replaced by the corresponding protein sequence from the B. licheniformis enzyme without affecting the functional behavior of the protein. Therefore, it is proposed that the amino acid residues which determine the functional characteristics of class A beta-lactamases should lie in between residues 37-200. The data obtained should facilitate the design of new beta-lactam antibiotics that will bypass the defense mechanism of the pathogenic bacteria.
Description: xiii, 145 leaves : ill. ; 30 cm.
PolyU Library Call No.: [THS] LG51 .H577M ABCT 2000 Cheung
URI: http://hdl.handle.net/10397/2935
Rights: All rights reserved.
Appears in Collections:Thesis

Files in This Item:
File Description SizeFormat 
b15352584_link.htmFor PolyU Users 162 BHTMLView/Open
b15352584_ir.pdfFor All Users (Non-printable) 4.17 MBAdobe PDFView/Open
Show full item record

Page view(s)

684
Last Week
2
Last month
Checked on Apr 30, 2017

Download(s)

218
Checked on Apr 30, 2017

Google ScholarTM

Check



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.