RSC Advances p. 3910 - 3918 (2016)
Update date:2022-08-30
Topics:
Kalyani
Munk
Mikkelsen
Meyer
A new laccase gene (mrlac) from Meiothermus ruber DSM 1279 was successfully overexpressed to produce a laccase (Mrlac) in soluble form in Escherichia coli during simultaneous overexpression of a chaperone protein (GroEL/ES). Without the GroEL/ES protein, the Mrlac overexpressed in E. coli constituted a huge amount of the total cellular protein, but the enzyme was localized in the insoluble fraction with no activity in the soluble fraction. Co-expression of the Mrlac with the E. coli GroEL/ES drastically improved proper folding and expression of active Mrlac in the soluble fraction. Spectroscopic analysis of the purified enzyme by UV/visible and electron paramagnetic resonance spectroscopy confirmed that the Mrlac was a multicopper oxidase. The Mrlac had a molecular weight of ~50 kDa and exhibited activity towards the canonical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ), and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants Km and kcat were 27.3 μM and 325 min-1 on ABTS, 4.2 μM and 106 min-1 on SGZ, and 3.01 μM and 115 min-1 on 2,6-DMP, respectively. Maximal enzyme activity was achieved at 70°C with ABTS as substrate. In addition, Mrlac exhibited a half-life for deactivation at 70°C and 75°C of about 120 min and 67 min, respectively, indicating that the Mrlac is intrinsically thermostable. Finally, Mrlac was efficient in catalyzing the removal of 2,4-dichlorophene (DCP) in aqueous solution, a trait which makes the enzyme potentially useful for environmentally friendly applications.
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