Protein engineering of laccase to enhance its activity and stability in the presence of organic solvents

Rasekh, Behnam; Khajeh, Khosro; Ranjbar, Bijan; Mollania, Nasrin; Almasinia, Banafsheh; Tirandaz, Hassan

doi:10.1002/elsc.201300042

Cited by 54 publications

(32 citation statements)

References 32 publications

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“…Incubation of the best mutant, 4‐OT A33I/FYA, for 10 h in the presence of 50 % ethanol resulted in only 25 % loss of activity, whereas the parental enzyme 4‐OT FYA lost all its activity upon incubation for 1 h in 10 % ethanol. 4‐OT A33I/FYA also showed an increase in thermostability compared to 4‐OT FYA, an effect that has also been observed for other enzymes that have been engineered towards increased solvent tolerance . We further show that 4‐OT A33I/FYA can be used to efficiently catalyze the Michael‐type addition of 3 to 4 a in the presence of 40 % ethanol, which permitted the use of a higher substrate loading (up to 15 m m 4 a ).…”

Section: Discussionsupporting

confidence: 71%

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

et al. 2020

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Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non‐aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4‐oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael‐type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its “Michaelase” activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The “hotspot” position Ala33 was then randomized in a highly enantioselective, but ethanol‐sensitive 4‐OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ‐nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability‐landscape‐guided enzyme engineering for efficient biocatalysis in non‐aqueous solvents.

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Section: Discussionsupporting

confidence: 71%

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

et al. 2020

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“…Similarly, the catalytic activity of laccase in S. carnis CPF-05 was almost lost when 10% of the organic solvents added (Olajuyigbe and Fatokun, 2017). In addition, the solvent tolerance of the enzyme is considered to be positively correlated with the thermal stability, which is also in line with the thermoactive and solvent tolerance of Lac 37 II in this study (Rasekh et al, 2014).…”

Section: Effect Of Organic Solvents On Activity Of Lac 37 IIsupporting

confidence: 78%

A Thermo-Active Laccase Isoenzyme From Trametes trogii and Its Potential for Dye Decolorization at High Temperature

Yang

et al. 2020

Front. Microbiol.

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A thermo-activation and thermostable laccase isoenzyme (Lac 37 II) produced by Trametes trogii S0301 at 37 • C was purified to apparent homogeneity by anionic exchange chromatography and sephadex G-75 chromatography, with 12.3% of yeiled and a specific activity of 343.1 U mg −1 . The molecular weight of the purified Lac 37 II was estimated to be approximately 56 kDa in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for the protein was 2.7 and 60 • C, respectively. The purified Lac 37 II showed higher resistance to all tested metal ions and organic solvents except for Fe 2+ and Cd 2+ at 37 • C and the activity of the purified Lac 37 was significantly enhanced by Cu 2+ at 50 mM. The K cat , K m , and K cat /K m of Lac 37 II were 2.977 s −1 , 16.1 µM, and 184.9 s −1 µM −1 , respecively, in the condition of pH 2.7 and 60 • C using ABTS as a substrate. Peptide-mass fingerprinting analysis showed that the Lac 37 II matched to the gene-deduced sequences of lcc3 in T. trogii BAFC 463, other than Lcc1, Lcc 2, and Lcc 4. Compared with laccase prepared at 28 • C, the onset of thermo-activation of Lac 37 II activity occurred at 30 • C with an increase of 10%, and reached its maximum at the temperatures range of 40-60 • C with an increase of about 40% of their original activity. Furthermore, Lac 37 II showed the efficient decolorization ability toward triphenylmethane dyes at 60 • C, with decolorization rates of 100 and 99.1% for 25 mg L −1 malachite and crystal violet in 5 h, respectively, when hydroxybenzotriazole (HBT) was used as a mediator. In conclusion, it is the first time to report a thermoactivation laccase from a thermophilic T. trogii strain, which has a better enzyme property and higher decolorization ability among fungal laccases, and it also has a further application prospective in the field of biotechnology.

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“…As the potential application of industrial biocatalysts would be greatly expanded if the enzyme could function at high temperatures and non-natural environments, therefore the enzyme activity and stability was studied in the presence of few organic solvents [15]. Due to the replacement of conventional organic solvents by ionic liquids here the experimental combination of rational and random protein engineering approaches has been successfully applied for enzyme modification to evaluate its performance in these solvents.…”

Section: Resultsmentioning

confidence: 99%

“…1), therefore substitution with any kinds of amino acids (charged, hydrophobic or polar) could affect the stability due to either electrostatic, hydrophobic, -, cation-or anion-interactions. It has already been shown in previous studies that replacing Glu 188 with hydrophobic (I, A) and positive charged (K) residues enhances the thermal stability and organic solvent tolerance of the enzyme [14,15]. Since it was not clear which residue(s) would be the optimal replacements for Glu 188 , we decided to use site saturation mutagenesis/screening.…”

Section: Mutation Designmentioning

confidence: 98%

A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions

Dabirmanesh

Khajeh

Ghazi

et al. 2015

International Journal of Biological Macromolecules

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Protein engineering of laccase to enhance its activity and stability in the presence of organic solvents

Cited by 54 publications

References 32 publications

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

A Thermo-Active Laccase Isoenzyme From Trametes trogii and Its Potential for Dye Decolorization at High Temperature

A semi-rational approach to obtain an ionic liquid tolerant bacterial laccase through π-type interactions

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