Aitao Li

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.

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.

Integrating interfacial self-assembly and electrostatic complexation at an aqueous interface for capsule synthesis and enzyme immobilization

A novel concept integrating supramolecular self-assembly and electrostatic complexation at an aqueous liquid–liquid interface to synthesize stable peptide–polymer hybrid capsules was developed. The concept was further applied for enzyme immobilization to give stable and active biocatalysts with low enzyme leakage and high encapsulation efficiency, enzyme loading, and recyclability.

Asymmetric trans-dihydroxylation of cyclic olefins by enzymatic or chemo-enzymatic sequential epoxidation and hydrolysis in one-pot

Novel and efficient one-pot enzymatic and chemo-enzymatic synthetic methods are developed for the asymmetric trans-dihydroxylations of cyclic olefins 1a and 1bvia sequential epoxidation and hydrolysis. The Novozym 435®-mediated epoxidation of cyclohexene 1a and subsequent hydrolysis of the intermediate cyclohexene oxide 2a with resting cells of Sphingomonas sp. HXN-200 in one-pot gave (1R,2R)-cyclohexane diol 3a in 84% ee and 95% conversion. trans-Dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b with the same enzymatic system gave the corresponding (3R,4R)-N-benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 93% ee and 94% conversion. In the one-pot chemo-enzymatic system, epoxidation of N-benzyloxycarbonyl 3-pyrroline 1b by m-CPBA and subsequent hydrolysis of epoxide intermediate 2b with resting cells of Sphingomonas sp. HXN-200 gave the trans-diol (3R,4R)-3b in 92% ee and 94–97% conversion. While the trans-dihydroxylation of cyclohexene 1a to (1R,2R)-cyclohexane diol 3a is reported for the first time, the trans-dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b to (3R,4R)-3b with such an enzymatic or chemo-enzymatic system afforded a much higher product concentration than the same reaction with the system using a microorganism containing the two necessary enzymes. The developed one-pot enzymatic and chemo-enzymatic systems for the asymmetric trans-dihydroxylation of olefins are new, easy to prepare, adjust and operate, are high yielding, complementary to Sharpless asymmetric dihydroxylation and particularly useful for the asymmetric synthesis of cyclic trans-diols.

Temperature-responsive nanobiocatalysts with an upper critical solution temperature for high performance biotransformation and easy catalyst recycling: efficient hydrolysis of cellulose to glucose

The development of an immobilized enzyme for efficient biocatalysis and catalyst recycling is of great importance in cost-effective and green chemical synthesis, with the hydrolysis of cellulose to glucose for the utilization of lignocellulosic biomass as a prominent and challenging example. We developed a novel concept of engineering temperature-responsive nanobiocatalysts with an upper critical solution temperature (UCST) for efficient catalysis as a soluble catalyst at a temperature >UCST and for easy catalyst separation as an insoluble catalyst at a temperature UCST). The hydrolysis of insoluble cellulose, such as filter paper and pre-treated oil palm Empty Fruit Bunch (EFB, a waste biomass), with a mixture of the UCST-nanobiocatalysts containing cellulase and cellobiase at 50 °C reached the same catalytic performance as the free enzymes and gave 97% and 93% glucose yield at 2 wt% of cellulase loading, respectively. These catalytic performances are much better than any other known immobilized cellulases, due to the use of soluble catalysts. The catalysts were easily recovered at 4 °C and recycled to retain 71% and 73% productivity in the 8th and 6th reaction cycles for hydrolyzing filter paper and pre-treated EFB, respectively. The engineered UCST-nanobiocatalysts are potentially useful for the practical green hydrolysis of cellulose to glucose.