Optimization of immobilization conditions of Bacillus atrophaeus FSHM2 lipase on maleic copolymer coated amine-modified graphene oxide nanosheets and its application for valeric acid esterification

The thermoalkalophilic lipase from Bacillus atrophaeus (BaL) was immobilized onto amine-functionalized graphene oxide nanosheets coated with the poly (maleic anhydride-alt-1-octadecene) copolymer (GO-NH2-PMAO) and activated with glutaraldehyde as spacer arm through interfacial activation and subsequent multipoint covalent attachment. Experimental design method was applied for optimization of immobilization conditions including GO-NH2-PMAO concentration, buffer concentration, pH, sonication time, enzyme concentration, glutaraldehyde concentration, time, and temperature. The optimum specific activity of the immobilized BaL (105.95 ± 2.37 U/mg) reached at 5 mg/mL for GO-NH2-PMAO, 25 mM of buffer, pH 6.0, 60 min sonication time, 100 mM glutaraldehyde, 60 U/mL of enzyme, and 4 h of immobilization time at 25 °C, which was very close to the predicted amount (106.08 ± 1.42 U/mg). Maximum immobilization yield (81.35%) and efficiency (277.63%) were determined in optimal immobilization conditions. The obtained results clearly indicated that the immobilized BaL exhibited better stability at extreme temperature and pH than the free BaL. At temperature of 90 °C and pH 11, more than 90% of the initial activity of the immobilized BaL was retained. Furthermore, the immobilized BaL retained about 90% of its initial activity after 10 days of storage and 6 cycles of application. The esterification studies showed that maximum bioconversion of valeric acid to pentyl valerate using the free BaL (34.5%) and the immobilized BaL (96.3%) occurred in the xylene medium after 48 h of incubation at 60 °C. Therefore, the BaL immobilized on GO-NH2-PMAO was introduced as an effective biocatalyst to synthesize green apple flavour ester.