Preparation of recombinant proteins
Genes encoding superfolder green fluorescent protein (sfGFP) and LipM7, a lipase recently discovered in our laboratory via metagenomics analysis (Table S2, to be published elsewhere) were cloned into the plasmid pET21a (Novagen, USA) between the NdeI and XhoI restriction enzyme sites to generate pET21a-sfGFP and pET21a-LipM7, respectively. For construction of plasmids encoding N-terminal phasin fusion proteins (PhaP-sfGFP and PhaP-LipM7), phasin gene phaP from Aeromonas hydrophila (accession number, UniprotKB-O32470) was synthesized by GenScript (China) and inserted between the HindIII and XhoI sites of pET21a-sfGFP or pET21a-LipM7. To prepare recombinant proteins fused with phasin at the C-terminus (sfGFP-PhaP and LipM7-PhaP), sfGFP and LipM7 genes were first cloned into the pET21a vector between the NdeI and HindIII sites, followed by insertion of the phasin sequence between the HindIII and XhoI sites (see Table S3 for primers used for plasmid construction).
Recombinant proteins were prepared from cultures of Escherichia coli strain BL21 (DE3) (Novagen, Germany) transformed with corresponding plasmids. Cells were grown at 37 °C in a 2 L baffled flask containing 400 mL of Luria-Bertani medium supplemented with 50 µg/mL of ampicillin. When the absorbance at 600 nm (OD600) reached 0.5 to 0.6, the culture broth was supplemented with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and incubated at 20 °C for a further 20 h. Cells were harvested by centrifugation (6,800 × g, 20 min) and lysed by sonication in 10 mL of 50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl, 10 mM imidazole, and 1 mg/mL lysozyme. The supernatant of the centrifuged lysate (6,800 × g, 20 min) was loaded onto a column containing 2 mL Ni-NTA agarose resin (Qiagen, Germany). After extensive washing with 20 mM imidazole in 50 mM sodium phosphate buffer, resin-bound proteins were eluted with 250 mM imidazole solution (1 mM imidazole for sfGFP-PhaP and PhaP-sfGFP). The eluate was dialyzed against 100 mL of 20 mM sodium phosphate buffer (pH 7.4). After adjusting to a protein concentration of 0.5 mg/mL, the protein solutions were stored at 4 °C prior to use.
The purity of proteins was analyzed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sizes of resolved proteins were estimated using PM2700 Protein Markers (SMOBIO, Taiwan). Protein concentration was determined by the Bradford assay following the manufacturer’s protocol (BioRad, USA). Fluorescence of sfGFP, sfGFP-PhaP, and PhaP-sfGFP was measured using a VICTOR X2 plate reader (Perkin Elmer, Waltham, MA, USA) set at wavelengths of 485 and 535 nm for excitation and emission, respectively. The enzymatic activity of LipM7 lipase was determined by a colorimetric method using p-nitrophenyl decanoate (pNPD) as a substrate, as described previously (Choi et al., 2013).
Fabrication of PHB nanofibers by electrospinning
PHB granules were dissolved in a 7:3 mixture of 2,2,2-trifluoroethanol and chloroform to 10% (w/v). The polymeric solution was transferred into a syringe and delivered to the needle at a flow rate of 0.5 mL/h. A constant voltage of 10 kV was applied to induce fiber formation. For preparation of nanofibers in two-dimensional (2D) sheet form, electrospun fibers were collected on aluminum foil connected to the ground. Three-dimensional (3D) nanofibrous sponges were prepared by collecting nanofibers in a coagulation bath filled with a 6:4 mixture of t-butanol and water. Prepared PHB nanofibers were dried in vacuum for 24 h, and stored in a desiccator prior to use.
Immobilization of proteins
PHB nanofibers (5 mg), 150 mg of PHB granules (Goodfellow, UK), 25 mg of Duolite A568 (Novozymes, Denmark), or 25 mg of Sipernat D17 (Degussa, Germany) were suspended 1 mL of protein solutions (0.2−0.3 mg) and incubated overnight at 4 °C or for 1 h at 30 °C under constant shaking. These supports were recovered by a brief centrifugation step and washed three times with 50 mM TRIS-HCl buffer (pH 8.0). The amount of protein adsorbed to the support was calculated by measuring the protein concentration in the buffer solution sampled before and after the immobilization procedures.
Characterization of lipase immobilization on PHB supports
Different concentrations of LipM7-PhaP dissolved in 10 mM sodium phosphate (pH 7.4) were immobilized on PHB nanofibers and PHB granules (average diameter: 2.5 µm). The amount of LipM7-PhaP immobilized on the PHB support was estimated by quantifying unbound proteins. The maximum loading capacity (qmax) and the dissociation constant (kd) were determined by applying the protein concentration to the Langmuir isotherm model.
Analysis of transesterification reactions by immobilized LipM7-PhaP
PHB nanofibers containing immobilized LipM7-PhaP (100 mg) were added to a 10 mL solution of 1 M octanoic acid and 1 M MeOH. The reaction mixture was incubated at 30 °C under constant orbital shaking at 250 rpm. Samples (100 µL) were withdrawn every 12 h until the reaction reached equilibrium. Withdrawn samples were mixed with 900 µL of chloroform containing n-hexane as an internal standard. Synthesized methyl octanoate, and residual octanoic acid and methanol were analyzed by gas chromatography on an HP-5 capillary column (30 m × 0.32 mm × 0.25 µm, J&W Scientific, Agilent, CA) equipped with a flame ionization detector (FID). Helium was used as the carrier gas at a flow rate of 1.3 mL/min. The temperatures of the injector and detector were set at 250 °C and 340 °C, respectively. Starting from 100 °C, the oven temperature was sequentially raised to 260 °C (15 °C/min) and 315 °C (5 °C/min), and held at this temperature for 15 min. Samples (1 µL) were injected in the split mode.