title: A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli.
abstract: 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 ∼20mg/L of bacterial culture, showing significant improvement over the laccase produced in E.coli BL21 strain under aerobic condition. The specific activity of 13U/mg for purified laccase produced in micro-aerobic condition was higher than that of 1.07U/mg observed for the purified enzyme obtained in aerobic condition in Origami. The kinetic Km and kcat parameters for laccase-induced oxidation reactions were 46μM and 23s(-1) for ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and 19.6μM and 24s(-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.
authors.affiliation: Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Azam
authors.initials: A
authors.lastname: Safary
authors.affiliation: Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran; Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran. Electronic address: moniri@kaums.ac.ir.
authors.forename: Rezvan
authors.initials: R
authors.lastname: Moniri
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Hamzeh-Mivehroud
authors.affiliation: Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Electronic address: dastmalchi.s@tbzmed.ac.ir.
authors.forename: Siavoush
authors.initials: S
authors.lastname: Dastmalchi
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects*; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D006219:Halogens / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D006219:Halogens; D011994:Recombinant Proteins; D012997:Solvents; D042845:Laccase
keywords: 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (PubChem CID: 5464076); Bacillus sp. SL-1; Cytoplasmic expression; EDTA (PubChem CID: 6049); Inclusion body; Micro-aerobic expression; Multicopper oxidase; NaN(3) (PubChem CID: 33557); Origami B (DE3); SDS (PubChem CID: 3423265); Sodium Chloride (PubChem CID: 5234); Syringaldazine (PubChem CID: 5379425)