Enzyme-Polymer Hybrid Nanocatalyst Prepared by Microemulsion Polymerization

Supporting information for

Enzyme-polymer Hybrid Nanocatalyst Prepared by Microemulsion Polymerization

Minglan Yaoa, Xiufu Hua b,c,* a School of Food Science and Engineering, Nanjing University of Finance & Economics, Nanjing,

210046, 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: [email protected], TEL.: 86-10-6279-2750

Materials. Lipase from Candida rugosa (Type VII), p-nitrophenylbutyrate(p-NPB), p-nitrophenyl palmitate (p-NPP), Triton X-100, N-acryloxysuccinimide (NAS), sodium bis-2-ethylhexylsulfosuccinate (AOT), polyoxyethylene-4-lauryl ether (Brij 30), acrylamide (AM), N-isopropylacrylamide (NIPAM), N,N,N',N'- tetramethylethylenediamine (TEMED), ammonium persulfate (APS), hexane, N,N’- methylenebisacrylamide, tris(2,2’-bipyridine)ruthenium dichloride hexahydrate

2+ (Ru(bpy)3 ) were purchased from Sigma-Aldrich, U.S.A.

Preparation of microemulsion. A given amount of AOT and Brij 30 surfactant mixture was dispersed in hexane, followed by slowly adding phosphate buffer (pH 7.0

10 mM) containing monomer and enzyme to reach the defined W0 ([water]/

1 [surfactants])=10 under vigorously stirring at 30 °C. Then the suspension was kept for

20 minutes to obtain a macroscopically homogeneous solution.

Figure S1. The pseudoternary phase diagram of the surfactant (AOT/Brij 30, 1:3, w/w)

/aqueous/hexane system at 30 °C. The aqueous phase contains lipase (1 mg/mL) and AM/MBA (50 mg/mL, 5 mg/mL) or NIPAM/MBA (100 mg/mL, 5 mg/mL).

Acryloylation of lipase. To generate the vinyl groups on the lipase surface for the subsequent polymerization to chemically encapsulate lipase into polymer nanoparticles, a step of acryloylation of lipase was carried out as follows: 24 mg of N- acryloxysuccinimide (NAS) was dissolved in 1 mL of dimethyl sulfoxide and slowly added to 10 mL of the enzyme solution, with the molar ratio of NAS to lipase being

200:1. After reaction at 30 °C for 6 h, the solution was collected and dialyzed against phosphate buffer (50 mM, pH 7.0) at 4 °C for 24 h to remove unreacted reagents. The hydrolytic activity of modified lipase was determined using p-nitrophenyl palmitate (p-

NPP) as the substrate.

In situ polymerization in the inverse microemulsion. For a representative procedure, the W/O microemulsion was composed of 40 mL (70 w/w %) of hexane,

7.68 g (20 w/w %) of surfactant mixture of AOT and Brij 30 (mass ratio is 1:3), and 4 mL (10 w/w %) of aqueous phase (10 mM, pH 7.0 phosphate buffer) containing

2 acryloylated lipase (1 mg/mL), monomer (50 mg/mL AM, or 100 mg/mL NIPAM), cross-linker (5 mg/mL MBA), initiator (4 mg/mL APS). After the W/O microemulsion was incubated at 30 °C and purged with N2 for 30 min, the polymerization accelerator

TEMED (100 μL) was added, followed by the reaction at 30 °C for 2 h. After the reaction, hexane was evaporated under reduced pressure at 30 °C, resulting in a viscous product. The viscous product was washed with 60 mL of isopropyl alcohol and the suspension was centrifuged at 10000 rpm at 4 °C for 20 min. Finally, the precipitated solid was dissolved in 1 mL of deionized water and followed by dialysis against deionized water for 10 days at 4 °C to remove the surfactants.

Determination of the enzyme nanoparticles. The sizes of the lipase nanoparticles were analyzed by size exclusion chromatography using TSK-GEL SW4000xL column

(TOSHO) at 25 °C. The elution buffer was 0.1 M, pH 6.7 sodium phosphate containing

0.1 M Na2SO4 and 0.05% NaN3 at a flow rate of 0.5 mL/min. TEM of the lipase nanoparticle was observed using Hitachi JEOL200CX high resolution transmission electron microscope. Carbon-coated grids were prepared by adding a drop of sample solution, removing the excess and subjected to TEM measurement.

3 Figure S2. Size exclusion chromatography of native and nanoparticle encapsulated lipase. (Please see Table 1 in the main text for the detailed compositions of nanoparticle A and B)

Steady-state fluorescence spectrum. The fluorescence study was carried out using

-5 2+ 1.5×10 M of fluorescence probe, Ru(bpy)3 in the microemulsion (instrument: RF-

5301 PC, SHIMADZU). The organic phase was hexane, while the aqueous phase was

10 mM, pH 7.0 phosphate buffer containing AM/MBA (50 mg/mL, 5 mg/mL) or

NIPAM/MBA (100 mg/mL, 5 mg/mL). The total concentration of AOT and Brij 30 in the microemulsion was maintained at 440 mM while the W0 ([water]/[surfactants]) value was kept at 10 for all microemulsions with different AOT/ Brij ratios. The excitation wavelength was 460 nm, and the emission spectra were observed in the range of 500-650 nm. The excitation and emission slit width for fluorescence experiments were kept constant at 5 and 5 nm, respectively.

Activity of lipase in microemulsion. To all the microemulsions prepared for the steady-state fluorescence measurement, 10 μL of lipase solution (protein concentration:

0.1 mg/mL) was injected to 1 mL of the prepared microemulsion, and the suspension was vortexed for 1 minute to obtain a macroscopically homogeneous solution. Then 10

μL of p-nitrophenylbutyrate (p-NPB) in acetonitrile with the concentration of 1 M was injected to the microemulsion, followed by measuring the absorbance at 333 nm at 30

°C using a SHIMADZU MultiSpec-1501 UV-vis spectrometer.

Protein concentration and lipase activity assay. The lipase concentration was determined by bicinchoninic acid colorimetric protein assay (BCA) calibrated with bovine serum albumin (BSA). For the comparison of activities of native lipase, acryloylated lipase, and lipase encapsulated in polymer nanoparticles, p-nitrophenyl palmitate (p-NPP) was firstly dissolved in acetone and then diluted with phosphate buffer (50 mM, pH 7.0) containing 1.25%(w/v) Triton X-100, giving the final

4 concentration of 0.5 mM. The reaction was started by adding 50 μL sample containing

50 μg/mL of enzyme to 950 μL substrate solution and detected at 348 nm. For the measurement of activity of lipase encapsulated in PNIPAM nanoparticle at different temperatures, p-nitrophenyl acetate (p-NPA) was firstly dissolved in acetone at a concentration of 20 mM and then injected into the reaction media (50 mM, pH 7.0, phosphate buffer) containing enzyme nanoparticles, giving a final concentration of 0.5 mM. The increase of absorbance at 410 nm was then recorded.