Optimization and in Silico Analysis of a Cold-Adapted Lipase from an Antarctic Pseudomonas sp. Strain AMS8 Reaction in Triton X-100 Reverse Micelles

Abstract

A moderate yield of a purified enzyme can be achieved by using the simple technique of reverse micellar extraction (RME). RME is a liquid-liquid extraction method that uses a surfactant and an organic solvent to extract biomolecules. Instead of traditional chromatographic purification methods, which are tedious and expensive, RME using the nonionic surfactant Triton X-100 and toluene is used as an alternative purification technique to purify a recombinant cold-adapted lipase, AMS8. Various process parameters were optimized to maximize the activity recovery of the AMS8 lipase. The optimal conditions were found to be 50 mM sodium phosphate buffer, pH 7, 0.125 M NaCl, and 0.07 M Triton X-100 in toluene at 10 • C. Approximately 56% of the lipase activity was successfully recovered. Structural analysis of the lipase in a reverse micelle (RM) was performed using an in silico approach. The predicted model of AMS8 lipase was simulated in the Triton X-100/toluene reverse micelles from 5 to 40 • C. The lid 2 was slightly opened at 10 • C. However, the secondary structure of AMS8 was most affected in the non-catalytic domain compared to the catalytic domain, with an increased coil conformation. These results suggest that an AMS8 lipase can be extracted using Triton X-100/water/toluene micelles at low temperature. This RME approach will be an important tool for the downstream processing of recombinant cold-adapted lipases.2 of 15 absence of co-factors [6][7][8][9]. Cold-active lipases cover a broad spectrum of biotechnological applications, such as additives in detergents (cold washing), an additive in food industries (fermentation, cheese manufacture, bakery, meat tenderizing), bioremediations (Digesters, composting, oil, or xenobiotic biology applications), and molecular biology applications [10][11][12].Several conventional purification methods for cold-adapted lipases have been applied for many years in industrial and laboratory procedures [13,14]. For example, the lipase KE38 from Pseudomonas fluorescens KE38 was successfully purified using (NH 4 ) 2 SO 4 precipitation, Sephadex G-100 and, lastly, ultrafiltration with a recovery of 54.99% and a 41.13-fold purification [15]. Likewise, three purification steps were used to purify extracellular LipP from Pseudomonas sp. strain BII-I by using 20-65% (NH 4 ) 2 SO 4 precipitation followed by a diethylaminoethyl (DEAE)-cellulose and graphite column with 38-fold purification and a recovery of 17% of the lipase activity [13]. Although a good yield of purified enzyme was obtained from these chromatographic techniques, the production and purification of large amounts of enzyme are often laborious and expensive, which hinders applications for these enzymes [16]. Therefore, a standard method that enables the purification of large amounts of highly pure, active, and stable enzymes is urgently needed. Reverse micellar extraction is one of the alternative purification methods under consideration for continuous downstream processing of bulk enzymes.A reverse micelle (RM) is a surfactant...

A Broad Temperature Active Lipase Purified From a Psychrotrophic Bacterium of Sikkim Himalaya With Potential Application in Detergent Formulation

“…The 3D structure was significantly valid and supported by well-known parameters like ProSA web server, Ramachandran plot and Q mean score. Previously, homology modeling approach has been used successfully to predict the 3D structure of several cold-active lipases produced by psychrotrophic bacteria (De Pascale et al, 2008;Xu et al, 2010;Kamarudin et al, 2014;Jalil et al, 2018). The final 3D model obtained was further exploited for finding the active site, docking and MD simulation studies.…”

Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8

“…In order to further analyze the presence of Ca 2+ ions in the AMS8 lipase predicted structure, we used the database to explore the calcium-binding site of each of the Ca 2+ ions. The ligplot of the interaction involving the calcium-binding site of analysis from the pictorial database of 3D structures in the Protein Data Bank (PDBsum) was visualized using YASARA software [12] Figure 1 shows that the Ca1 was bound with Asn 284 (2.294 Å) and Glu 253 (2.209 Å) ( Figure 1b), Ca2 with Asp 283 (2.328 Å) and Thr 281 (2.381 Å) ( Figure 1c), Ca3 with Asp 378 (2.236 Å), Gly 391 (2.227 Å), Lys 393 (2.237 Å) and Gly 376 (2.272 Å) ( Figure 1d), Ca4 with Asp 387 (2.270 Å), Gly 383 (2.341 Å) and Gly 385 (2.310 Å) ( Figure 1e), Ca5 with Gly 392 (2.344 Å) and Gly 394 (2.339 Å) ( Figure 1f) and Ca6 with Phe 413 (2.178 Å) and Asp 416 (1.523 Å) ( Figure 1g). Both Ca1 and Ca2 were located at the catalytic domain, while Ca3, Ca4 and Ca5 were located at the RTX parallel β-roll motif repeat structure in the non-catalytic domain and Ca6 was also located at the non-catalytic domain of the AMS8 lipase structure.…”

“…The expression of AMS8 lipase was conducted as mentioned by Jalil et al with slight modifications [12]. The E. coli strain BL21(DE3) with recombinant plasmid pET32b/lipAMS8 was induced with IPTG (0.1 mM) for AMS8 lipase production at 20 • C with shaking at 180× g for 18 h. The production of AMS8 lipase was qualified by streaking the culture on an agar plate containing tributyrin 1% (v/v) and ampicillin (50 µg/mL).…”

The effect of single CNTs/GNPs and complexes on promoting the interfacial catalytic activity of lipase in conventional emulsions

“…Water‐in‐oil (W/O) microemulsions (also called reverse micelles) are a good microenvironment for enhancing the solubility of lipophilic substrates of lipases and have been widely studied due to their resemblance to biological membranes and their capability of solubilizing enzymes and catalyzing biochemical reactions . W/O microemulsions provide large aqueous–organic interfaces with very low interfacial tension, high thermodynamic stability, high local reaction concentration, high regioselectivity, high enzyme stability and low product inhibition in improving the efficiency of the lipase catalytic reaction …”