Biodiesel synthesis assisted by ultrasonication using engineered thermo-stable Proteus vulgaris lipase

Abstract

Nature has evolved and designed enzymes to perform an exquisite array of tasks, but in the pursuit of biotechnological interests, these enzymes must often be improved, altered, or even completely redesigned. In the present work, production of biodiesel was carried out using Neem oil and methanol catalysed by “engineered” Proteus vulgaris lipase (PVL). Two major issues have been addressed in this study in order to improve the efficiency of biodiesel synthesis by enzyme catalysis. The thermal stability of PVL was increased by introduction of a disulfide bond in G181 and T238 by mutation to cysteines. The transesterification reaction was carried out using sonication under different ultrasonic experimental conditions using a 20kHz horn. The results showed that the application of ultrasound, using 20kHz horn with 1cm tip diameter, decreased the reaction time from 22–24h to 30min at an applied power of 40W and methanol to oil molar ratio of 5:1. Temperature raised due to sonication had no effect on engineered PVL (PVLC181-238) activity. A comparative study of wild type (WT-PVL) and engineered PVLC181-238 for different temperature has been performed and results showed that introduction of a single disulfide in PVL significantly stabilized it, increasing the half-inactivation temperature (IT1/2) from 37°C for the WT-PVL to 50°C for the PVLC181-238 engineered one. In biodiesel synthesis also after immobilization on (Polysulfone) PS beads, PVLC181-238 showed better performance compared to WT-PVL.