n-Butyl levulinate (n-BL) has numerous applications in biofuel and green solvents. In this paper, we have reported mechanistic accounts of sonoenzymatic synthesis of n-BL using Novozym 435. Esterification parameters were optimized with statistical design of experiments. Application of 35 kHz sonication boosted n-BL yield from 70.9% to 92.22%. Mechanistic investigation using Ping-Pong Bi-Bi kinetics model and molecular docking simulations revealed interesting influence of sonication on esterification reaction. Exposure to sonication induced significant changes in the secondary structure of enzyme, as revealed in ATR-FTIR analysis. Sonication increased α-helix content of enzyme that increased enzyme activity by opening of flapping lid and widening the catalytic cavity. Sonication also caused unfolding of secondary structural motifs with rise in random coil content that favored formation of enzyme-ligand complexes. These effects enhanced reaction velocity and substrate affinity with reduction in inhibition and unfavorable dissociation of intermediate complexes, which ultimately enhanced n-BL yield.

In recent years, bacteria from genus Clostridia have attracted attention of research community because of their biofuel production capabilities. Present study reports comparative genomic (CG) analysis of 48 genomes of solventogenic and saccharolytic Clostridia. We have focused on central carbon metabolism and general stress response in the analysis. Comprehensive summaries on comparison of general genome features, COG categories, CDSs of the energy, catabolic, and sporulation pathways are given. Furthermore, we have proposed two new genome-scale metabolic (GSM) models iKK848 and iKK1425 for Clostridium pasteurianum DSM 525 = ATCC 6013 and Clostridium acetobutylicum ATCC 824, respectively. These GSM models are most comprehensive in that they account for the largest number of reactions, metabolites, and genes as compared to previous models. Model quality and metabolic flux optimization for biomass growth using iKK1425 and iKK848 are compared with previous literature. Our models had the highest quality score of 61% and 77%.

Ultrasound has emerged as an efficient green technology for intensification of lipase-catalyzed processes. These processes are of high significance in the context of renewable fuels synthesis. In this paper, we have attempted to reveal the molecular mechanism of sonication-induced enhancement of lipase-catalysed reactions. Using hybrid quantum mechanics/ molecular mechanics computations, we have initially determined the structure and amino acid composition of the binding pockets of two common lipase enzyme, viz. CALB and TLL. Next, these binding pockets were visualized using softwares PyMOL and VMD to deduce their location in different motifs of the secondary structure of lipases. Finally, the docking analysis of different ligands was performed to reveal the amino acid residues involved for each ligand and the nature of their interactions. Our results revealed that most of the binding pockets are located in the α-helix and random coil motifs of enzyme and binding interactions are of the type hydrogen bond and hydrophobic interactions. Previous literature reports significant rise in α-helix and random coil contents of lipases with sonication. This observation concurs with our analysis, and suggests rise in enzyme activity with sonication due to widening of catalytic cavity and easier accessibility to binding pockets. Thus, our analysis provides molecular-level insight into the sonication-induced enhancement in kinetics and yield of lipase-catalyzed reactions.