Efficient transformation of grease to biodiesel using highly active and easily recyclable magnetic nanobiocatalyst aggregates.

Green and efficient production of biodiesel (FAME) from waste grease containing high amount of free fatty acid (FFA) was achieved by using novel magnetic nanobiocatalyst aggregates (MNA). Thermomyces lanuginosus Lipase (TLL) and Candida antarctica Lipase B (CALB) were covalently immobilized on core-shell structured iron oxide magnetic nanoparticle (80 nm), respectively, followed by freeze-dry to give MNA (13-17 µm) with high yield (80-89%) and high enzyme loading (61 mg TLL or 22 mg CALB per gram MNA). MNA TL showed the best performance among immobilized enzymes known thus for the production of FAME from grease (17 wt.% FFA) with methanol, giving 99% yield in 12 h (3.3 wt.% catalyst). MNA TL was easily separated under magnetic field and reused, retaining 88% productivity in 11th cycle. MNA CA converted >97% FFA in grease (17 wt.% FFA) to FAME in 12 h (0.45 wt.% catalyst), being useful in two-step transformation of grease to biodiesel.

Efficient production of biodiesel from waste grease: one-pot esterification and transesterification with tandem lipases.

A novel concept and efficient method for producing biodiesel (FAME) from grease (15-40wt% free fatty acid, FFA) were developed by using tandem lipases for one-pot esterification of FFA and transesterification of triglyceride with methanol in a solvent-free system. Combining immobilized Candida antarctica lipase B (CALB) (Novozyme 435) favoring the esterification and immobilized Thermomyces lanuginosus lipase (TLL) (Lipozyme TLIM) preferring the transesterification at 2:8 (wt/wt) gave FAME in 80% yield, being better than that with Novozyme 435 or Lipozyme TLIM. Recombinant Escherichia coli (Calb/Tll) co-expressing CALB and TLL was engineered as a more efficient tandem-lipases system. Using wet or dry cells (4wt%) gave FAME in 87% or 95% yield, which is much better than that with E. coli cells expressing either CALB or TLL alone. Cells of E. coli (Calb/Tll) were recycled for five times and retained 75% productivity, thus being practical for producing biodiesel from grease.

Whole-cell based solvent-free system for one-pot production of biodiesel from waste grease.

A whole-cell based solvent-free system was developed for efficient conversion of waste grease to biodiesel via one-pot esterification and transesterification. By isolation and screening of lipase-producing strains from soil, Serratia marcescens YXJ-1002 was discovered for the biotransformation of grease to biodiesel. The lipase (SML) from this strain was cloned and expressed in Escherichia coli as an intracellular enzyme, showing 6 times higher whole-cell based hydrolysis activity than that of wild type strain. The recombinant cells were used for biodiesel production from waste grease in one-pot reactions containing no solvent with the addition of methanol in several small portions, and 97% yield of biodiesel (FAME) was achieved under optimized conditions. In addition, the whole-cell biocatalysts showed excellent reusability, retaining 74% productivity after 4 cycles. The developed system, biocatalyst, and process enable the efficient, low-cost, and green production of biodiesel from waste grease, providing with a potential industrial application.

Reversible clustering of magnetic nanobiocatalysts for high-performance biocatalysis and easy catalyst recycling.

Reversible clusters of nanobiocatalysts are developed via non-covalent interaction among enzyme-bound iron oxide magnetic nanoparticles. Dissociation of the clusters by shaking during biotransformation enables high catalytic performance, and re-clustering by stopping shaking after reaction allows for easy magnetic separation. The novel concept is demonstrated with alcohol dehydrogenase RDR for the enantioselective reduction of 7-methoxy-2-tetralone.