Ethanol as additive enhance the performance of immobilized lipase LipA from Pseudomonas aeruginosa on polypropylene support.

Immobilization is practical to upgrade enzymes, increasing their performance and expanding their applications. The recombinant, solvent tolerant lipase LipA PSA01 from Pseudomonas aeruginosa was immobilized on polypropylene Accurel® MP1004 to improve its performance. We investigated the effect of ethanol as an additive during the immobilization process at three concentrations (20%, 25%, and 30%) on the operational behavior of the enzyme. The immobilization efficiency was higher than 92%, and the immobilized enzymes showed hyperactivation and thermal resistance depending on the concentration of ethanol. For example, at 70 °C, the free enzyme lost the activity, while the prepared one with ethanol 25% conserved a residual activity of up to 73.3% (∆ T15 50 = 27.1 °C). LipA immobilized had an optimal pH value lower than that of the free enzyme, and the organic solvent tolerance of the immobilized enzymes depended on the ethanol used. Hence, the immobilized enzyme with ethanol 25% showed hyperactivation to more solvents than the soluble enzyme. Remarkable stability towards methanol (up to 8 folds) was evidenced in all the immobilized preparations. The immobilized enzyme changed their chemo preference, and it hydrolyzed oils preferentially with short-chain than those with long-chain. LipA had a notable shelf-life after one year, keeping its activity up to 87%. Ethanol facilitated the access of the enzyme to the hydrophobic support and increased its activity and stability according to the amount of ethanol added.

Functional Heterologous Expression of Mature Lipase LipA from Pseudomonas aeruginosa PSA01 in Escherichia coli SHuffle and BL21 (DE3): Effect of the Expression Host on Thermal Stability and Solvent Tolerance of the Enzyme Produced.

This study aimed to express heterologously the lipase LipA from Pseudomonas aeruginosa PSA01 obtained from palm fruit residues. In previous approaches, LipA was expressed in Escherichia coli fused with its signal peptide and without its disulfide bond, displaying low activity. We cloned the mature LipA with its truncated chaperone Lif in a dual plasmid and overexpressed the enzyme in two E. coli strains: the traditional BL21 (DE3) and the SHuffle® strain, engineered to produce stable cytoplasmic disulfide bonds. We evaluated the effect of the disulfide bond on LipA stability using molecular dynamics. We expressed LipA successfully under isopropyl ß-d-1-thio-galactopyranoside (IPTG) and slow autoinducing conditions. The SHuffle LipA showed higher residual activity at 45 °C and a greater hyperactivation after incubation with ethanol than the enzyme produced by E. coli BL21 (DE3). Conversely, the latter was slightly more stable in methanol 50% and 60% (t½: 49.5 min and 9 min) than the SHuffle LipA (t½: 31.5 min and 7.4 min). The molecular dynamics simulations showed that removing the disulfide bond caused some regions of LipA to become less flexible and some others to become more flexible, significantly affecting the closing lid and partially exposing the active site at all times.