Fig. 2

Expression of free γ-CGTase and identification of PHA-based nanobeads generated by co-expression of γ-CGTase and PhaC. a SDS–PAGE analysis of free γ-CGTase purified by the α-CD-Sepharose 6B affinity column. Lane M, protein ladder; Lane 1, the lytic supernatant of the recombinant E. coli BL21 (DE3) strain harboring the pET22b (+)-cgt plasmid after induction; Lane 2, resuspension of the precipitate obtained from the lytic supernatant (Lane 1) after adding 40% saturated ammonium sulfate solution; Lane 3, the protein solution of the resuspended precipitate (Lane 2) after desalting; Lanes 4 and 5, the flow-through of the desalting protein loaded onto the α-CD-Sepharose 6B affinity column; Lanes 6–8, the eluate obtained from the affinity column. b SDS–PAGE analysis of free γ-CGTase purified by size exclusion chromatography (SEC) equipped with a HiLoad 16/600 Superdex 200 pg column. Lane M, protein ladder; Lane 1, the purified free γ-CGTase eluted from the column. c–e Transmission electron microscopy analysis of unmodified E. coli cells (c), recombinant E. coli carrying the blank plasmid pCDFD-ABC (d), and recombinant E. coli carrying pCDFD-ABC-cgt (e) after induction. f SDS–PAGE analysis of γ-CGTase-PHA beads expressed by the recombinant E. coli BL21 (DE3) strain harboring the pCDFD-ABC-cgt vector. Lane M, protein ladder; Lane 1, non-induced strain; Lane 2, lytic supernatant of the IPTG-induced strain; Lane 3, lytic precipitation of the IPTG-induced strain. g Determination of the hydrolysis and cyclization activity of free γ-CGTase and immobilized γ-CGTase. Values are averages of results from triplicate trials; the error bars indicate the SD values