Six-membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent-free lipase-mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym (R) 435 (immobilized Candida antarctica lipase BCalBa.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by-productmethanolwas the most efficient setup. Higher conversion (81.6%) in the flow reaction compared to batch process (72%) was obtained using same amount of N435 (20% (w/w) N435:TMP) at 12 h, and the undesirable dimer and oligomer formation were suppressed. Moreover, the product was recovered easily without extra separation steps, and the biocatalyst and the molecular sieves remained intact for subsequent regeneration and recycling. The reaction of CalB with DMC and the primary transesterification product, monocarbonated TMP, respectively, as acyl donors was evaluated by in silico modeling and empirically to determine the role of the enzyme in the formation of cyclic carbonates and other side products. DMC was shown to be the preferred acyl donor, suggesting that TMP and its carbonated derivatives serve only as acyl acceptors in the lipase-catalyzed reaction. Subsequent cyclization to cyclic carbonate is catalyzed at increased temperature and not by the enzyme. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:375-382, 2017