Amicon stirred ultrafiltration cell

Throughout the present work, biochemical properties of an N-terminally truncated variant of the Thermus thermophilus HB27 E34Tt esterase (YP_004875.1), expressed by means of the Kluyveromyces lactis NRRL-Y1140 yeast strain [22 (link)], were studied. The recombinant strain obtained was then named KLEST-3S (expressing the ∆N16 variant, with an estimated molecular weight of 34.3 kDa). The strain producing KLEST-3S was cultivated without pH control at 30 °C and 250 rpm in YPL (1% yeast extract (w/v), 2% peptone (w/v) and 2% lactose (w/v)). After cultivation for 72 h, lipolytic activity was recovered in the cell-free culture media. Post-incubated cell-free medium was concentrated by dia-ultrafiltration using tangential flow filtration (TFF) cartridges with a 10 kDa cut-off polyethersulfone membrane (Millipore Corporation, Burlington, MA, USA). When required, this concentrated post-incubated medium was newly concentrated by using an Amicon stirred ultrafiltration cell (10 KDa cut-off) (Millipore Corporation). The concentrated liquid constituted the crude enzyme solution, essentially free of contaminant proteins, with which all the experiments were carried out. Reactivity studies in organic media were conducted with dry samples of KLEST-3S obtained by freeze-drying the enzyme solution.

The filtration of oligosaccharide extract was evaluated in an Amicon-stirred ultrafiltration cell (Millipore Corporation, Darmstadt, Germany) (MWCO of 30 kDa) at standard temperature and pressure (25 °C, 1 atm). Permeate and retained samples were collected, lyophilized, and stored in a desiccator at room temperature (25 °C).
Initial Oligosaccharide hydrolyzed extract were characterized by the DNS method and the total carbohydrate method (both using glucose as the calibration standard). In addition, the protein content was evaluated by the Kjeldhal method and minerals as its ash content (Table 2). After filtration, the fraction’s antiproliferative activity was determined, and the recovery yield was reported as % mg solid in the corresponding fraction/ mg total solid in the original extract (Table 3).

Outer membrane vesicles were prepared as described earlier (Klimentova et al., 2019 (link)) from large-scale cultivations (2–6 L). Briefly, bacteria from agar plates were inoculated in BHI for pre-cultivation (ca 20 h, 37°C, 200 rpm). After centrifugation (6,000 × g, 15 min, 25°C), the desired volume of suspension of OD₆₀₀ = 0.1 was prepared and cultivated for 14–16 h. Bacteria were pelleted (10,000 × g, 20 min, 4°C), and the supernatants were sterilized by filtration through a 0.22-mm vacuum-driven filter and concentrated using Amicon Stirred Ultrafiltration Cell (Millipore) through a membrane of regenerated cellulose with 100 kDa cutoff (Millipore). OMVs were pelleted (100,000 × g, 90 min at 4°C), resuspended in 45% OptiPrep (Sigma-Aldrich) in 10 mM HEPES/0.85% NaCl, pH 7.4 (HEPES buffer), and overlaid with a step OptiPrep gradient of 40–20%. The gradient was centrifuged (100,000 × g, overnight, 4°C) in a swinging bucket rotor. After centrifugation, the top fractions containing opaque white bands were collected, diluted 8× with HEPES buffer, and centrifuged (100,000 × g, 2 h, at 4°C). The supernatant was removed and the pellet was washed again to remove the residual OptiPrep. The final pellet was suspended in physiological saline and the protein concentration was determined with the Micro BCA™ Protein Assay Kit (Pierce).