Open AccessProtein engineering of laccase to enhance its activity and stability in the presence of organic solvents
Author(s) -
Rasekh Behnam,
Khajeh Khosro,
Ranjbar Bijan,
Mollania Nasrin,
Almasinia Banafsheh,
Tirandaz Hassan
Publication year - 2014
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.201300042
Subject(s) - chemistry , laccase , site directed mutagenesis , thermal stability , protein engineering , mutagenesis , solvent , bacillus subtilis , residue (chemistry) , enzyme , methanol , ethanol , alkyl , organic solvent , directed evolution , mutant , active site , organic chemistry , biochemistry , chemical engineering , bacteria , gene , engineering , genetics , biology
In our previous study, we reported an increase in the thermal stability of bacterial laccase from Bacillus sp. HR03 using site‐directed mutagenesis. Three‐dimensional model of this enzyme showed a negative patch in the connecting loop between domains 1 and 2. In the present study, the stability of laccase in organic solvents was improved by introducing nonpolar (E188 → A, I, L, and V) and positively charged (E188 → K and R) residues in this region by site‐directed mutagenesis. Irreversible thermoinactivation, C 50 value (organic solvent concentration at which 50% of enzyme activity remains), change in transition‐state stabilization energy, and kinetic parameters of the wild type and its variants were calculated in the presence and absence of various organic solvents (ethanol, methanol, and 1‐propanol). All variants showed higher C 50 values when compared to the wild type. Nonpolar amino acid substitutions were found to be the most efficient mutants for their remarkable increase in C 50 value and a decrease in thermoinactivation rate in the presence of mentioned solvents. Data showed that replacing a negative residue with hydrophobic residues on the surface of a protein could enhance thermoresistance as well as solvent stability. The stability of the resulting enzymes was dependent on the length of the alkyl chain. Results demonstrated that solvent tolerance was positively correlated with thermal stability.