The thermostable transketolase from Geobacillus stearothermophilus (TK gst) was successfully engineered for the synthesis of aliphatic acyloins with varying carbon backbone lengths (C 5 À C 10) based on protein structure-guided studies. Efficient TK gst variants were identified with enhanced activities for substrate combinations of aliphatic aldehydes as acceptors together with aliphatic pyruvate homologues as donors. The TK gst single variant L382F was able to catalyze efficiently the transfer of the ketol group from hydroxypyruvate on all targeted aliphatic aldehydes (C 3 À C 8) to give the corresponding 1,3-dihydroxy ketones with good yields and excellent enantioselectivity. The combination of the H102L/H474S mutation previously designed for the improved utilization of aliphatic pyruvate homologues together with a F435I exchange gave the new variant H102L/H474S/F435I, which is able to transfer the acyl goup of 2-oxobutyrate and 2-oxovalerate to aliphatic aldehydes, giving mono hydroxylated ketones.

The design of drug-loaded nanoparticles (NPs) appears to be a suitable strategy for the prolonged plasma concentration of therapeutic payloads, higher bioavailability, and the reduction of side effects compared with classical chemotherapies. In most cases, NPs are prepared from (co)polymers obtained through chemical polymerization. However, procedures have been developed to synthesize some polymers via enzymatic polymerization in the absence of chemical initiators. The aim of this work was to compare the acute in vitro cytotoxicities and cell uptake of NPs prepared from poly(benzyl malate) (PMLABe) synthesized by chemical and enzymatic polymerization. Herein, we report the synthesis and characterization of eight PMLABe-based polymers. Corresponding NPs were produced, their cytotoxicity was studied in hepatoma HepaRG cells, and their uptake by primary macrophages and HepaRG cells was measured. In vitro cell viability evidenced a mild toxicity of the NPs only at high concentrations/densities of NPs in culture media. These data did not evidence a higher biocompatibility of the NPs prepared from enzymatic polymerization, and further demonstrated that chemical polymerization and the nanoprecipitation procedure led to biocompatible PMLABe-based NPs. In contrast, NPs produced from enzymatically synthesized polymers were more efficiently internalized than NPs produced from chemically synthesized polymers. The efficient uptake, combined with low cytotoxicity, indicate that PMLABe-based NPs are suitable nanovectors for drug delivery, deserving further evaluation in vivo to target either hepatocytes or resident liver macrophages.

A c c e p t e d M a n u s c r i p t 2 catalytic histidine of the enzyme preferentially to serine, in the form of a strong hydrogen bond with one of the "acidic" protons of MLABe, thus supporting the important role of the catalytic histidine in the polymerization of such cyclic lactones.