Different Effects of Salt Bridges near the Active Site of Cold-Adapted Proteus mirabilis Lipase on Thermal and Organic Solvent Stabilities

Abstract

Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix η1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities.

Solvent Tolerance Improvement of Lipases Enhanced Their Applications: State of the Art

“…Many studies have identified newly formed salt bridges in lipase variants with improved organic solvent tolerance [ 122 , 123 , 129 ]. To investigate the contribution of salt bridges near the active site to the stability of Proteus mirabilis lipase (PML) in organic solvents, Dachuri et al [ 130 ] substituted alanine for two arginine residues (R237 and R241) and one aspartate residue (D248) which formed salt bridges near the active site and then evaluated their efficiency in 10–40% ( v / v ) DMSO and 10–40% ( v / v ) methanol. The results showed that the double mutant (R237A/D248A) even lost its activity completely within 1 h in the 40% organic solvent, whereas the wild type had about 80% activity in both 40% ( v / v ) organic solvents.…”