Supporting information for

Production of Biodiesel Using a Nanoscaled Immobilized Lipase as the Catalyst

Yulong Liua, Xiufu Hua b,c,*

a Department of Basic Teaching, Yancheng Institute of Technology, Yancheng, 224051, China

b Department of Scientific Research and Development, Tsinghua University, Beijing ,100084, China

c Nanjing Ruhan Biotech Co., Ltd. Nanjing, 210012, China

*Corresponding author

Department of Scientific Research and Development, Tsinghua University, Beijing, 100084,China. E-mail address: , TEL.: 86-10-6279-2750

Materials.Lipase NS81006 from genetically modified Aspergillus niger was purchased from Novo Industries (Denmark). p-Nitrophenyl palmitate (p-NPP), acrylamide (AM), N-acryloxysuccinimide (NAS), ammonium persulfate (APS), N,N,N´,N´-tetramethylethylenediamine (TEMED), methanol and hexane were purchased from Sigma-Aldrich, USA.

Hydrolytic Activity Assay.p-NPP was dissolved in acetone and diluted with phosphate buffer (50 mM, pH 7.0) containing 1.25% (w/v) Triton X-100 to give a final concentration of 0.5 mM.[1] The reaction was started by adding 50 L of enzyme solution to 950 L of the substrate solution and the absorbance was monitored at 348 nm. For triglyceride hydrolysis, 1 U of lipase activity is the amount of lipase that produces 1 mol of free fatty acid per minute under the given assay condition. The protein concentration was determined by bicinchoninic acid colorimetric assay.

Synthesis of Lipase Nanogel.Lipase was reacted with NAS to generate vinyl groups on the enzyme surface. To determine the optimal pH for this reaction, lipase NS81006 was dissolved in 20 mL of phosphate buffer (50 mM, pH 7.0 or pH 6.0) or acetate buffer (50 mM, pH 5.0 or pH 4.0) at a protein concentration of 2 mg/mL, followed by a 24-h dialysis against the same buffer. To determine the optimal NAS/lipase ratio, NAS dissolved in dimethyl sulfoxide was added to 5 mL of the enzyme solution to yield a concentration of 10, 20, 30 or 40 mM. After the reaction at 30 °C for 5 h, the sample was dialyzed against phosphate buffer (50 mM, pH 7.0) at 4 °C for 24 h to remove unreacted reagents. For in-situ polymerization to prepare lipase nanogel, acrylamide was dissolved in acryloylated lipase solution at a concentration of 25, 50, 70 or 100 mg/mL. After 30 min of N2 purging, the polymerization was initiated by adding 1 mg/mL of ammonium persulfate and 33 µM of TEMED followed by reaction at 30 °C for 12 h. The sample was ultrafiltered (molecular weight cutoff 100 000) using a Microcon centrifugal filter device (Millipore) to remove free lipase and then dialyzed against phosphate buffer (50 mM, pH 7.0) at 4 °C for 24 h to remove unreacted reagents.

Size Exclusion Chromatography.Size exclusion chromatography was conducted using a TSK-GEL SW2000xL column (Tosho) and a Shimadzu RF-10AxL fluorescence detector. The mobile phase was 0.1 M sodium phosphate (pH 6.7) containing 0.1 M Na2SO4 at a flow rate of 0.5 mL/min. The eluate was monitored using excitation and emission wavelengths of 280 and 340 nm, respectively.

Transmission Electron Microscopy.TEM images of the samples were recorded using a Hitachi JEOL200CX high-resolution transmission electron microscope. Carbon-coated grids were prepared by adding a drop of sample solution, removing the excess and applying 1% sodium phosphotungstate (pH 7.0).

Lipase-Catalyzed Production of Biodiesel.The enzymatic reaction was carried out in a 250 mL flask in a mechanically agitated thermostatic water bath. A mixture of 100 g of soybean oil, 10 g of water, 30 mL of methanol and 1000 U of lipase or lipase nanogel was emulsified by stirring at 800 rpm. Samples of 1.5 mL taken from the reaction mixture at specified times were centrifuged and the upper layerswere subjected to gas chromatography analysis.

Determination of Methyl Ester Yield.Methyl esters were analyzed using a GC-14B gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a CP-FFAP CB capillary column (25 m × 0.32 mm × 0.30 m) supplied by Agilent. A sample of 5 L was precisely measured and mixed thoroughly with 600 L of 0.7 mM heptadecanoic acid methyl ester (internal standard). The column temperature was kept at 180 °C for 0.5 min, heated to 250 °C at 10 °C/min, and then maintained for 6 min. The injector and detector were set at 245 °C and 250 °C, respectively.

References

[1]López, N.; Pernas, M. A.; Pastrana, L. M.; Sánchez, A.; Valero, F.; Rúa, M. L. Biotechnol. Prog.2004, 20, 65.

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