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

• Published in: Journal of biotechnology 2016-06, Vol.227, p.56-63
• Main Authors: Safary, Azam, Moniri, Rezvan, Hamzeh-Mivehroud, Maryam, Dastmalchi, Siavoush
• Format: Article
• Language: English
• Subjects: Adaptation, Physiological - drug effects; Bacillus; Bacillus - enzymology; Bacillus sp. SL-1; Bacteria; Base Sequence; Blotting, Western; Cytoplasmic expression; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Enzyme Stability - drug effects; Enzymes; Escherichia coli; Escherichia coli - genetics; Halogens - pharmacology; Hydrogen-Ion Concentration; Inclusion body; Kinetics; Laccase; Laccase - genetics; Laccase - isolation & purification; Laccase - metabolism; Micro-aerobic expression; Multicopper oxidase; Origami B (DE3); Recombinant; Recombinant Proteins - isolation & purification; Solubility; Solvents; Spectrophotometry, Ultraviolet; Strain; Temperature
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2016.04.006

•Laccase gene from newly isolated Bacillus sp. SL-1 was cloned and overexpressed.•A method was introduced for soluble expression of laccase in Origami strain.•The laccase activity under different oxygen availability was investigated.•The produced laccase retained 50% activity after one hour incubation at 70°C.•The enzyme was resistant to various inhibitors and organic solvents. 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.