Extremophilic lipases for industrial applications: A general review.

The combination of covalent and ionic exchange immobilizations enables the coimmobilization on vinyl sulfone activated supports and the reuse of the most stable immobilized enzyme

In this study, a Taguchi experimental design was used for optimzing the immobilization of the lipase Eversa® Transform 2.0 (EVS) onto a hybrid support consisting of chitosan (CHI) and agarose (AGA), with glutaraldehyde (GLU) used as the support activator. The biocatalyst obtained was characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetry (TGA), Energy Dispersive Spectroscopy (EDS), and Scanning Electron Microscopy (SEM). The optimized reaction conditions (60 min, 5 mM ionic strength, 1% GLU concentration, and 5 mg protein load per g of support) resulted in a highly active biocatalyst (74.39 ± 0.48 U/g) and delivered an immobilization yield of 74.20 ± 0.28%. The biocatalyst produced was observed to lose only 15.3% of its activity after 61 days of storage. The activity was also observed to increase by 96.70% ± 0.76, 27.34% ± 2.34, and 84.35% ± 1.68 in the presence of the organic solvents hexane, cyclohexane, and methanol, respectively. Additionally, the byocatalist showed more pronounced activity at temperatures above 50 °C and was still able to retain approximately 30% of it at 70 °C. These values were found to be higher at alkaline pHs, as the activity of Eversa® 2.0 Transform saw an increase of up to 140% at pH 9. The desorption tests performed did not reveal any enzymatic detachment from the support. The novel biocatalyst also showed promising ester-lubricating properties. Furthermore, the in silico study revealed a binding affinity of −5.1 kcal/mol between oleic acid and the enzyme, suggesting that the combination of the substrate and the lipase was more stable and therefore, suitable for esterification. 

A novel hybrid biocatalyst from immobilized Eversa® Transform 2.0 lipase and its application in biolubricant synthesis

This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.

https://www.mdpi.com/2073-4344/12/9/1065/pdf?version=1663643028

A Convenient U-Shape Microreactor for Continuous Flow Biocatalysis with Enzyme-Coated Magnetic Nanoparticles-Lipase-Catalyzed Enantiomer Selective Acylation of 4-(Morpholin-4-yl)butan-2-ol

https://doi.org/10.1016/j.bios.2022.114032

Hamdy Kashtoh

Papers

Temperature-resistant and solvent-tolerant lipases as industrial biocatalysts: Biotechnological approaches and applications.