Effect of sub‐ and super‐critical CO₂ pretreatment on conformation and catalytic properties evaluation of two commercial enzymes of CALB and Lipase PS

Abstract

BACKGROUND: It is vitally important to understand the effect of sub-/super-critical CO 2 pretreatment on enzyme conformation and its activity, which should improve the understanding of enzymatic biotransformation applications in nonaqueous media. This study evaluated the effect of sub-/super-critical CO 2 treatment, including pressure (6 and 10 MPa), exposure time (20, 30 and 150 min) and temperature (35 and 40 • C), on the conformation (e.g. 1D, 2D and 3D structures) and catalytic properties (e.g. residual activity, kinetics constants (K m and V max ), activation energies (E α ), thermo-stability and organic solvent tolerance) of two commercial enzymes, CALB and lipase PS in their solution forms.RESULTS: Compared with the blank control, the 1D structure of both treated lipases was unchanged, whereas their 2D and 3D structures were altered to some extent. The highest relative activities were 105% and 116% for CALB and lipase PS, respectively. For CALB, V max /K m value significantly increased, while for lipase PS, V max /K m value was almost constant. Both treated enzymes showed high thermo-stability with recovery of enzyme activity up to 76% and 80%, respectively. Also, both treated enzymes presented high organic solvent tolerance. CONCLUSIONS: It was speculated that 2D, 3D structure alterations were probably responsible for the satisfactory catalytic properties of enzymes treated with sub-/super-critical CO 2 .

Enzymatic activity and conformational and morphological studies of four commercial lipases treated with supercritical carbon dioxide

“…The enzyme activity of free enzymes after SC-CO 2 treatment was determined as the initial rate in the hydrolysis reaction of the olive oil triglycerides [12]. In a typical assay, 1 mL of enzyme preparation was added to the substrate, consisting on 4 mL of 10 % homogenized olive oil and 5 mL of 50 mM phosphate buffer pH = 7.0.…”

“…It has been also proposed that SC-CO 2 may interact with hydrophobic tryptophan (Trp) and tyrosine (Tyr) residues in the enzyme structure during the treatment [10][11][12][13][14]. Liu et al [11] proposed that, below a pressure threshold that depends on the enzyme nature and on the experimental conditions, CO 2 can interact with Trp and Tyr residues by means of weak interactions, promoting conformational changes that lead to a more accessible active site.…”

“…Changes in the conformational structure due to enzyme residues and SC-CO 2 interactions [10][11][12][13][14], and/or extraction of water and impurities from the enzyme preparation [15] could explain this enzyme activity enhancement.…”

Activity of immobilized lipase from Candida antarctica (Lipozyme 435) and its performance on the esterification of oleic acid in supercritical carbon dioxide

“…Investigating the influence of temperature on the activity of two commercial immobilized lipases submitted to supercritical CO 2 , Oliveira et al [14] observed that at higher temperatures, up to 70 °C, the activity reductions were at least 8%. Liu et al [13] evaluated the effect of sub and supercritical CO 2 treatment, including pressure, exposure time and temperature on the residual activity of two commercial enzymes (Candida antarctica Lipase B (CALB) and lipase PS in solution). Differently from this work, the authors observed an increase of activity for both lipases after treatment with sub-and supercritical CO 2 , in which the highest residual activities obtained were 105% and 116% for CALB and lipase PS, respectively, at the same conditions (10 MPa, 40 °C and 30 minutes of exposure time).…”

“…The enzymatic activity was measured through the volumetric method, which is based on the titrimetric determination of the free acids released from triacylglycerols by lipase catalyzed hydrolysis [12,13]. About 5 ml of olive oil emulsion, water and arabic gum (7%), 2 ml of phosphate buffer pH 7.0 (0.1M) and 10 mg of enzyme were incubated for one hour in a shaker incubator (TE-421, Tecnal, Piracicaba-SP/Brazil) at 40 °C and 140 orbitals per minute (OPM).…”