fulltext.study @t Gmail

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
17090 42640 2013 6 PDF Available
Title
An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli
Abstract

•In vitro refolding of recombinant bacterial laccase from Bacillus HR03 is studied.•We have shown that the drop dilution as a powerful method for refolding of laccase.•Differences in kinetic parameters of soluble and refolded enzyme were also observed.•Structural changes during the refolding monitored using fluorescence spectroscopy

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO4 and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.

Keywords
Laccase; Refolding; Inclusion body; Intrinsic fluorescence; Overexpression
First Page Preview
An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 52, Issues 6–7, 10 May 2013, Pages 325–330
Authors
, , , , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering