A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli

Safary, A. and Moniri, R. and Hamzeh-Mivehroud, M. and Dastmalchi, S. (2016) A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli. Journal of Biotechnology, 227. pp. 56-63.

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An efficient method was introduced for soluble expression of recombinant laccase (rpCotA(SL-1)) from a newly isolated halo-thermotolerant Bacillus sp. SL-1 in modified Escherichia coli, trxB2/gor2 mutant (Origami� B (DE3)). The yield of purified soluble laccase in Origami strain under micro-aerobic condition was ~20 mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13 U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07 U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46 μM and 23 s-1 for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6 μM and 24 s-1 for SGZ (syringaldazine) substrates, respectively. The rpCotA(SL-1) displayed thermostability at 70 °C and tolerance to specified concentrations of NaCl, NaN3, EDTA and SDS as inhibitors. The enzyme was relatively stable in the presence of different concentration of organic solvents, however the residual activity was adversely affected as the dipole moment of the solvents increase. Here we successfully report the production of soluble and functional laccase in Origami at the expression level suitable for industrial application. © 2016 Elsevier B.V.

Item Type: Article
Additional Information: cited By 0
Uncontrolled Keywords: Bacteriology; Escherichia coli; Purification, Bacillus sp; Cytoplasmic expression; Inclusion bodies; Micro aerobics; Multicopper oxidase; Origami B (DE3), Enzymes, bacterial enzyme; laccase; ribosome DNA, Article; Bacillus; bacterial genome; bacterium culture; biochemical analysis; dipole; enzyme activity; enzyme analysis; enzyme synthesis; Escherichia coli; gene expression system; gene sequence; molecular weight; nonhuman; oxidation; priority journal; protein expression; room temperature; sequence alignment; sequence analysis; temperature dependence; thermostability
Subjects: Biochemistry, Genetics and Molecular Biology
Divisions: Faculty of Medicine > Basic Sciences > Department of Anatomy
Depositing User: editor . 2
Date Deposited: 05 Mar 2017 23:12
Last Modified: 05 Mar 2017 23:12
URI: http://eprints.kaums.ac.ir/id/eprint/126

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