2.6.1 Visualization of extracellular enzyme activity on plates
2.6.1.1 α-Amylase (AmyQ)
Single colonies of the B. subtilis strains carrying plasmid pHCMC04, pNDH15, pNDH16, pNDH19, pNDH20, pNDH21, pNDH22, pNDH37-amyQ, pNDH89, pNDH90 and pNDH91 were grown for 24 h on LB plates containing 1 mM IPTG or 1% xylose and 2% insoluble starch and stained with I2/KI solution [105]. Pictures were recorded using a digital camera.
2.6.1.2 Cellulase (CelA and CelB)
Single colonies of the B. subtilis strains carrying plasmid pNDH37-celA or pNDH37-celB were grown for 24 h on LB plates containing 1 mM IPTG and 0.5% CMC and stained with 1% congo red solution [22]. Pictures were taken by a digital camera.
2.6.2 Measurement of the β-galactosidase activity
2.6.2.1 β-Galactosidase BgaB
Blue colonies from LB-Xgal plates were used for determination of β-galactosidase activity (white colonies for the strains with pHCMC01-bgaB). B. subtilis strains IHA01-Spac-BgaB and 1012 carrying pHCMC01-bgaB, pHCMC02-bgaB, pHCMC03-bgaB, pHCMC04-bgaB, pHCMC05-bgaB and pNDH33-bgaB were grown and samples collected as described under 2.3.3 and 2.5.7. The activity was determined at 55 0C as described [99] with the exception that the BgaB activity was measured in a microtiterplate reader (VersaMax, Molecular Devices).
One unit is defined as ∆A420*OD578-1*min-1 and displayed as units/OD578 for all the results except for the result using pHCMCs plasmid series which were displayed as units/mg protein, in which one OD578 is defined as the optical density of the samples used in the assay, A420 is the absorbance of the samples measured by the microtiterplate reader and min indicates the incubation time of the plate at 55 0C.
2.6.2.2 β-Galactosidase LacZ
B. subtilis strain 1012 carrying pHCMC03-lacZ and NDH20, NDH21 containing the transcriptional fusions PyhcS-lacZ and PywpE-lacZ were grown and samples collected according to section 2.3.3. β-Galactosidase activity were measured at 405 nm at 28 0C as describe elsewhere [168]. One unit is defined as Vmax*OD578-1, in which OD578 is defined as the optical density of the samples used in the assay and Vmax is the maximum kinetic rate reported as miliOD/minute. The data were displayed as units/mg protein for pHCMC03-lacZ and units/OD578 for strains NDH20 and NDH21.
2.6.3 Measurement of the α-amylase activity
Strains NDH03 with different plasmids (pHCMC04, pNDH15, pNDH16, pNDH19, pNDH20, pNDH21, pNDH22) were grown in LB medium at 37 0C as described under 2.3.3. Cells were grown to an OD578 0.1 - 0.2 at 37 0C, then 1 mM of IPTG was added to induce production of sortase A. When the cultures reached an OD578 of 0.8, 0.5% xylose was added to induce production of wild-type or hybrid α-amylase. All cultures were further grown for 2 h. Then, aliquots were collected and the cells were separated from the growth medium by
centrifugation. Samples were kept at - 20 0C until determination of the enzymatic activity.
Proteins released from the cell walls were prepared as described in section 2.5.3.
Strain 1012 carrying plasmid pNDH37-amyQ was grown in LB medium with Cm at 37 0C and samples were collected as described under 2.3.3. Culture supernatants were withdrawn after centrifugation step. Samples were kept at - 20 0C.
α-Amylase activity was determined as described [105]. Samples of cells were transferred into 1.5-ml Eppendorf tubes and samples from supernatants into microtiterplates. α-Amylase activities were determined with whole cells and with the supernatants and presented in units per OD578. One unit is defined as a decrease in OD620 of 0.1. All experiments were repeated at least twice.
2.6.4 Determination of covalent anchor-reporters on the cell wall
Colonies exhibiting halos on plate containing insoluble starch were used for this experiment.
Strains WW02 and NDH03 harbouring pNDH15 or pNDH16 were grown as described in section 2.6.3 and preparation of proteins released from the cell wall was carried out as described in section 2.5.3. Protein loading buffer was added directly to the culture supernatant. These samples were applied for immunoblot analysis using the antibodies against α-amylase (AmyQ). The same protocol was applied for other B. subtilis strains carrying plasmids pNDH15, pNDH16, pNDH19, pNDH20, pNDH21, pNDH22, pNDH89 and pNDH90.
Colonies from strain NDH03/pNDH18 and WW02/pNDH18 exhibiting green fluorescence under the microscope (2.7.2) on LB plates containing 1% xylose were used for this experiment. These strains were grown as described in section 2.6.3 and preparation of proteins released from the cell wall according to section 2.5.3. Protein loading buffer was added directly to the culture supernatant. These samples were applied for immunoblot analysis using the antibodies against GFP.
2.6.5 Determination of the number of α-amylase molecules on the cell surface
Cells from two different cultures were withdrawn, sedimented by centrifugation, washed twice with the PB and treated with lysozyme to release the α-amylase as described in 2.5.3.
Cells were centrifuged, and 5 µl of supernatant corresponding to 850,000 cells were applied
per lane. α-Amylase from two different cultures was analysed. Defined amounts of purified α-amylase (Sigma) from 2.5 to 20 ng (corresponding to 0.05 - 0.4 pmol) were run on the same gel. The collected pictures were analysed by Quantity One (BioRad). Only the material in the upper band of the samples was quantified (Fig. 3.15).
2.6.6 Kinetics of α-amylase immobilization on the cell wall
Strain NDH03 with a plasmids able to express α-amylase with a 94-aa and 123-aa spacer (pNDH16 and pNDH19, respectively) were prepared and the α-amylase activity was determined as in section 2.6.3. Cells were withdrawn for the determination of the α-amylase activity at the time points indicated in Fig. 3.18. Before collecting the samples, tetracycline was added at a final concentration of 20 µg/ ml to inhibit protein synthesis, and the cultures were shaken for another 10 min and then stored in ice. Proteins released from the cell walls were prepared as described in section 2.5.3.