Introducing a covalent thiol-based protected immobilized acetylcholinesterase with enhanced enzymatic performances for biosynthesis of esters

Abstract

Acetylcholinesterase covalent immobilization was performed on mercaptoacetic acid-functionalized magnetic nanoparticles. Upon the thiol-based protection of the enzyme active center, its kinetics, stability, and activity in organic solvents were enhanced and its initial activity was fully recovered. The optimal results for the immobilization (i.e., yield=98.3 ± 5.2 %; enzyme loading=1.20 ± 0.06 mg mg−1) were found at an acetylcholinesterase initial concentration of 12.0 mg mL−1 and an immobilization time of 4.0 hrs. The optimum pH of both enzymes was over 7.0–9.0. The optimal temperature was measured as 40.0–45.0 and 30 ºC for immobilized and free AChE, respectively. The immobilized enzyme retained about 92.1 ± 3.0 % and 91.5 ± 3.0 % of the initial activity after 65 days and 10 cycles, respectively. The Km and Vmax were calculated as 136.2 ± 8.2 µM and 4.3 ± 0.2 µmol mg−1 min−1for the immobilized and 162.0 ± 9.7 µM and 3.63 ± 0.20 µmol mg−1 min−1for the free enzyme. The novel biocatalyst was utilized for ultra-fast esterification, for the first time, revealing 73.0 ± 2.3 % yield after 30.0 min and 85.0 ± 2.7 % after 15.0 min along with fully saving the catalytic efficiency over 6 and 8 cycles for solvent-free and solvent-controlled reactions, respectively. As a consequence, this biocatalyst is an excellent candidate for the application of the AChE enzyme in the commercial synthesis of esters.