Expression plasmid and chemicals
Construction of the recombinant plasmid cgt/pET-20b(+), which directs expression of the wild-type α-CGTase from P. macerans strain JFB05-01 (CCTCC M203062) fused to the pelB signal peptide, has been described in a previous report (Li et al. 2009). Peptone and yeast extract powder were obtained from Oxoid (Basingstoke, Hampshire, United Kingdom). Isopropyl β-d-1-thiogalactopyranoside (IPTG), O-nitrophenyl-β-d-galactopyranoside (ONPG) and N-phenyl-α-naphthylamine (NPN) were purchased from Beyotime Institute of Biotechnology (Nantong, China). Glycerin and methyl orange were purchased from Shanghai Chemical Reagent Ltd. (Shanghai, China). All inorganic compounds were of reagent grade or higher quality.
A single colony of E. coli BL21 (DE3) harboring plasmid cgt/pET-20b(+) was used to inoculate 50 mL of Luria–Bertani (LB) medium supplemented with 100 mg/mL ampicillin (inoculum size, approximately 0.1%). This starter culture was incubated on a rotary shaker (200 rpm) at 37 °C until the optical density at 600 nm (OD600) reached 0.6 (about 8 h). The resulting culture was diluted (1:25) into 100 mL of terrific broth medium in a 500-mL flask, and IPTG was added to a final concentration of 0.01 mM to induce protein expression. The induction was allowed to proceed on a rotary shaker (200 rpm) at the specified temperature for 90 h. Samples of the culture were taken at intervals and analyzed for cell concentration and enzyme activity.
Cell fractionation was performed as previously described, with minor modifications (Li et al. 2010a, b). A 1-mL sample of the culture solution was centrifuged at 10,000 rpm for 10 min and the supernatant was collected. To separate the periplasmic fraction, the bacterial pellet from the 1-mL sample was washed twice with pure water and then completely resuspended in pure water containing 25% (w/v) sucrose and 1 mM EDTA. This suspension was adjusted to a final volume of 1 L, incubated on ice for 2 h, and then centrifuged at 10,000 rpm for 5 min. The supernatant was collected as the periplasmic fraction. The pellet was resuspended in l mL of 10 mM sodium phosphate buffer (pH 6.2) containing 0.5 mM calcium chloride and disrupted by ultrasonication with a sonifier (Branson, USA) for 5 min. After centrifugation at 10,000 rpm for 10 min, the residual cell fragments were mixed with 100 μL of 1% (w/v) SDS-PAGE loading buffer and heated for 10 min in a boiling water bath. After a final centrifugation, the α-CGTase inclusion bodies were in the upper buffer.
α-CGTase activity assay
α-CGTase activity was determined using the methyl orange method (Li et al. 2013a, b). The culture supernatant (0.1 mL) was mixed with 0.9 mL of 5% (w/v) soluble starch in 50 mM phosphate buffer (pH 6.0) and incubated at 40 °C for 10 min. After terminating the reaction by the addition of 1.0 mL HCl (1.0 M), 1.0 mL of 0.1 mM methyl orange in 50 mM phosphate buffer (pH 6.0) was added. After the mixture had reacted at 16 °C for 20 min, the amount of α-cyclodextrin in the mixture was determined by measuring the absorbance at 505 nm. One unit of α-cyclodextrin-forming activity was defined as the amount of enzyme able to produce 1 µmol of cyclodextrin per min.
Analysis of inner and outer membrane permeability
Samples were removed from the fermentation specified times after induction and centrifuged at 10,000 rpm for 10 min. The cell pellets were washed twice with 10 mM sodium phosphate buffer (pH 7.4) and diluted with the same buffer until the OD600 reached 0.5. Samples (1 mL) of these cell suspensions were used to assess the permeability of their inner and outer membranes as described below.
A previously described absorbance assay was used to evaluate the permeability of the inner membrane (Liao et al. 2004). Briefly, cell samples described above were mixed with ONPG (100 μg/mL) to assess permeability of the inner membrane. Cleavage of the ONPG that entered the cell, which is catalyzed by cytosolic β-galactosidase, was determined by measuring the absorption of light at 420 nm using a spectrophotometer. Measurements were taken every 5 or 10 min for 2 h.
Completeness of the outer membrane was assessed using a previously described NPN fluorescence assay (Eriksson et al. 2002). The fluorescence of NPN is weaker in aqueous solution than it is in hydrophobic environments. When NPN is applied to intact cells, it is excluded from the cells’ interior by the lipopolysaccharide layer of the cells’ outer membranes. Once the outer membrane is compromised, NPN gains access to the lipid bilayer and its fluorescence becomes strong in this hydrophobic environment. Cell suspensions (1 mL, prepared as described in the previous paragraph) were treated with NPN at a final concentration of 10 mM. Fluorescence was measured every 5 or 10 min for 2 h using excitation and emission wavelengths of 350 and 428 nm, respectively, and slit widths of 1 nm. Elevated NPN fluorescence was considered evidence of compromised cell membranes.
All measurements were performed in triplicate. The mean and standard deviations of the data collected were calculated using SPSS 17.0 software (SPSS Incorporated, Chicago, Illinois, USA).