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.

Rheology of milk foams produced by steam injection.

UNLABELLED: Rheology of milk foams generated by steam injection was studied during the transient destabilization process using steady flow and dynamic oscillatory techniques: yield stress (τ(y) ) values were obtained from a stress ramp (0.2 to 25 Pa) and from strain amplitude sweep (0.001 to 3 at 1 Hz of frequency); elastic (G') and viscous (G″) moduli were measured by frequency sweep (0.1 to 10 Hz at 0.05 of strain); and the apparent viscosity (η(a) ) was obtained from the flow curves generated from the stress ramp. The effect of plate roughness and the sweep time on τ(y) was also assessed. Yield stress was found to increase with plate roughness whereas it decreased with the sweep time. The values of yield stress and moduli-G' and G″-increased during foam destabilization as a consequence of the changes in foam properties, especially the gas volume fraction, ϕ, and bubble size, R(32) (Sauter mean bubble radius). Thus, a relationship between τ(y) , ϕ, R(32) , and σ(surface tension) was established. The changes in the apparent viscosity, η, showed that the foams behaved like a shear thinning fluid beyond the yield point, fitting the modified Cross model with the relaxation time parameter (λ) also depending on the gas volume fraction. Overall, it was concluded that the viscoelastic behavior of the foam below the yield point and liquid-like behavior thereafter both vary during destabilization due to changes in the foam characteristics. PRACTICAL APPLICATION: Studying the transient rheology of milk foams during destabilization contributes to our knowledge of the relationships between the changes in foam properties: texture and mouth feel during the consumption of hot foamed beverages.