Effects of Lid 1 Mutagenesis on Lid Displacement, Catalytic Performances and Thermostability of Cold-active Pseudomonas AMS8 Lipase in Toluene

Abstract

Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37 °C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of k cat /K m in 0.5% (v/v) toluene, mainly with p NPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for p NPL and p NPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35 °C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.

Chemoenzymatic Synthesis of New Aromatic Esters of Mono- and Oligosaccharides

“…The great challenge of the lipase-catalyzed esterification of sugars with phenolic acids is the solubility of sugars in highly polar organic solvents in which lipases can be denaturated or inactivated [25,26], mainly because polar solvents can penetrate the active site of enzyme, leading to protein unfolding [27]. The possibility of tuning the lipase catalytic activity or organic solvent stability by protein engineering represents a very promising approach [28,29] but is usually targeting a specific application and the mutant enzymes are commercially unavailable. Therefore, sugar acetals and alkyl derivatives are mostly used to overcome the solubility impediment.…”

Mutation of a distal gating residue modulates NADH binding in NADH:Quinone oxidoreductase from Pseudomonas aeruginosa PAO1

“…For successful protein engineering, mutation of gating residues must not be detrimental to protein function ( 9 , 10 , 11 , 12 , 13 ). Three considerations about gating residues support the idea of their attractiveness for protein engineering ( 1 , 12 , 16 , 17 , 18 ). First, the mutation of a gating residue is generally not detrimental to protein function, as gating residues are often spatially separated from the enzyme's active site.…”