We developed a nanoparticulate Rhizopus arrhizus lipase formulation to enhance its activity and to increase the conversion yield of lipids into fatty acid methyl esters (FAME, a.k.a., biodiesel). More than 95% purity of the lipase was achieved in a two-step purification. Nanoparticle formulation was afforded by co-lyophilization of the lipase with methyl-βcyclodextrin (MβCD), an established lyoprotectant. Scanning electron microscopy and dynamic light scattering measurements showed a size of 75− 200 nm for the nanoparticles depending on the ratio of lipase-to-MβCD employed during co-lyophilization. Fourier transform infrared spectroscopic analysis by Gaussian curve fitting of the resolution-enhanced amide I region of lyophilized and nanoparticulate lipase indicated a more native-like secondary structure in the latter. A 98% substrate-to-FAME conversion was achieved in 10 h in n-hexane by lipase nanoparticles, whereas the crude and lyophilized enzyme showed 65 and 70% conversion in 18 h, respectively. In this aspect, the lipase nanoparticles were superior to all other reported systems. Operational stability after 5 catalytic conversions of nanoparticles was found to be >81%. In summary, we herein developed a novel lipase formulation for efficient catalysis in lipid-to-biodiesel conversion.

rugosa lipase nanoparticles as robust catalyst for biodiesel production in organic solvents.Inexpensive but resourceful sources of lipids, for example, used cooking oil (UCO) and brown grease (BG), which often contain large amounts of free fatty acids (FFA), are difficult to convert into biodiesel economically and in good yield. Candida rugosa lipase nanoparticles (cNP) were formed first and subsequently cross-linked nanoparticles (CLNP) were obtained by crosslinking of them. Alternatively, cNP were conjugated to magnetic nanoparticles (mNP) to achieve a cNP-mNP conjugate. All three formulations were employed in three different organic solvents (n-heptane, 1,4-dioxane, and t-butanol) to produce biodiesel using BG and UCO in the transesterification reaction with ethanol and methanol. The radii of nanoparticles (NP) were 5.5, 75, 100, 85 nm for mNP, cNP, CLNP, and cNP-mNP, respectively, as measured by scanning/transmission electron microscopy and dynamic light scattering. The catalytic efficiency (Kcat/KM) of cNP, CLNP, and cNP-mNP was increased ca. -25, -68, -176 folds in n-heptane and -35, -131, -262 folds in 1,4-dioxane compared to the lyophilized lipase in the model transesterification reaction of p-nitrophenyl palmitate (PNPP) with ethanol. In biodiesel formation, the best performance with 100% conversion of BG was achieved under optimum conditions with cNP-mNP, ethanol at a 1:3 molar ratio of lipid-to-alcohol, NP at a 1:0.1 weight ratio of lipid-to-enzyme, and water at a 1:0.04 weight ratio of enzyme-to-water at 30 o C for 35 h. The operational stability of the CLNP and cNP-mNP was sustained even after five consequent biodiesel batch conversions while 50% and 82% residual activity (storage stability) were retained after 40 d.