Pruebas y certificación de los puntos de red instalados

2.5.1 Optical density

Optical densities were measured at 600nm. Samples were diluted with RO water to give a maximum absorbance of 0.8. The absorbance was read against blank media at the same dilution.

2.5.2 Dry cell weights

E. coli dry cell weights were measured in two ways. In the first method, a known volume of whole broth was aliquoted onto a pre-dried 0.2pm filter and vacuum filtered (Whatman International Ltd, Maidstone, Kent, UK). It was then dried to constant

weight at 105®C in an oven for 24 h and then weighed. The second method, using microcentrifiige tubes, was used for samples that were too viscous or of very high cell weight (above 40 g L'*). Microcentrifuge tubes were dried to constant weight in a similar way to the filters. A 1 mL aliquot of culture was pipetted into the microcentrifuge tube (Griener Labortechnik, Dursley, UK) and centrifuged for 10 min at 11,500 X g (Biofuge 13, Heraeus Instruments, Brentwood, UK). The resulting supernatant was decanted and the cells resuspended in an equal volume of RO water. The cells were further centrifuged for 10 min, the supernatant decanted, and the tube then dried for 24 h or until constant weight was achieved.

S. lividans dry cell weights were carried out by aliquoting 5 mL of whole broth onto a pre-dried AP25 pre-filter (Millipore Ltd, Watford, UK) and drying to constant weight at 60°C.

2.5.3 Plasmid stability

Plasmid stability was determined by measuring the viable cell count on a selective plate in comparison with the viability on a non-selective plate. A serial dilution of cells was carried out fi’om approximately 10'^ to 10"* in 0.7% sodium chloride solution (Oxoid). Duplicate aliquots of 0.1 mL of a serial dilution ranging from 10'^ to 10'^ were dropped onto selective and non-selective plates. The liquid was then spread over the surface of each plate using a flame sterilised spreader. The plates were then incubated overnight at 37®C. Plasmid stabilities were calculated by comparing the viable colonies on kanamycin plates with the total number of colonies on kanamycin free plates.

2.5.4 Whole cell fractionation

E. coli; Whole broth samples (1 mL) were collected in 1.6 mL microfuge tubes (Griener Labortechnik) and harvested by centrifugation at 11,500 x g. The supernatant (referred to as the extracellular fraction), was removed and the cell pellets were resuspended in 0.4 mL buffer containing 20% (w/v) sucrose, 0.5 mg mL'* hen egg white lysozyme (EC 3.2.1.17, L6876, Sigma) and 1 mM EDTA and statically incubated for 10 min, at which point 0.4 mL of RO water was added to the mixture and further incubated for 10 min. The cells were harvested by centrifugation for 10 min (periplasmic fraction). The

remaining cell pellet was resuspended in 50 mM Tris/HCl (pH 7.5) and sonicated with one 30s burst at 8 micron amplitudes (Soniprep 150, MSE Scientific instruments Ltd, Crawley, UK) followed by centrifugation for 10 min at 11,500 x g.

S. lividans; Whole broth samples were collected in 1 mL microfuge tubes (Griener Labortechnik) at various time intervals and spun down at 11,500 x g to give the media supernatant. This was assayed for total protein and a-amylase.

2.6 Analyses & Assays

2.6.1 a-Amylase assay

a-Amylase activity was measured using an assay modified by French (1993) based on the method of Blanchin-Roland & Masson (1989) in which activity was determined by measuring the rate of decrease of a coloured starch/Ij complex. This method was adapted for use on a microtitre plate. The substrate used was 0.5% (w/v) soluble starch (Sigma) in 15 mM sodium phosphate buffer at pH 5.8 which had previously been heated to boiling point and filtered through No 1 filter paper (Whatman International Ltd) whilst hot. The iodine stop reagent was freshly prepared by adding 0.2 mL of stock solution (2.2% (w/v) iodine/4.4% (w/v) KI) to 100 mL of 2% (w/v) potassium iodide solution. An appropriate dilution of the periplasmic fraction was made up to 0.15 mL using 15 mM phosphate buffer in a microtitre plate and pre-incubated at 50°C. At the beginning of the assay 0.15 mL of the pre-incubated starch solution was added to the sample wells. At various times thereafter, 0.015 mL of the reaction mixture was pipetted into 0.3 mL of lodine/KI stop solution on a separate microtitre plate. At the end of the assay the plate was measured at 620nm using a microtitre plate reader (MR7000 Dynatech, Billingshurst, West Sussex, UK).

2.6.2 P-cyclodextrin assay

This method was adapted fi’om Makela ei al, (1987) for the determination of coupled P- cyclodextrin on affinity supports. Standards of known concentration were made up and 0.5 mL of each concentration was aliquotted into 4 mL cuvettes. 0.25 mL of IM sodium

carbonate solution was then added and the total volume made up to 2.3 mL. 0.2 mL of phenolphthalein (Sigma P9750) working solution (1:10 dilution of 3.75 mM phenolphthalein in 94%(v/v) ethanol) was added to the mixture. The cuvettes were then read at 553nm. The assay could also be carried out on microtitre plates where the input volumes were decreased by a factor of 10 and the plate read at 560nm on a microtitre plate reader (MR7000, Dynatech, Billingshurst, West Sussex, UK)

2.6.3 Lysozyme assay

Lysozyme activity was determined by measuring lytic activity against Micrococcus lysodeikticus cells (Sigma) based on the method used by Locquet et al (1968). M

lysodeikticus (25 mg) was added to 100 mL of 0.067 M sodium phosphate buffer (pH 6.2). 0.9 mL of this buffer was then pipetted into a 1 cm cuvette and a 0.1 mL sample was added. The cuvette was mixed by inversion and measured using a time drive function at 450nm over 1 minute (Uvikon 922 spectrophotometer, Kontron Instruments, Watford, UK). Lysozyme values were calculated against a known set of lysozyme standards over the range 0-4000 U mL'*, allowing the lysozyme activity to be calculated. One unit of activity is defined as a change in absorbance of 0.001 at 450nm over 1 min. at constant temperature using a 1 mL suspension of 0.25 mg mL'* M lysodeikticus cells.

2.6.4 Glucose-6-phosphate dehydrogenase (G6PDH) assay

G6PDH assays were performed using the method of Deutsch (1983), in which the rate of appearance of NADPH is measured at 340nm. The assay mixture was made up as follows; Tris(hydroxymethyl)amino methane (86 mM); MgCb (6.9 mM); glucose-6- phosphate (1 mM); and NADP^ (0.39 mM) at pH 7.4. 0.015 mL of suitably diluted sample and 0.3 mL of assay mixture were added to a microtitre plate and the change in absorbance measured at 340nm was measured for 15 min using time drive kinetics software (Biolinx, Dynatech, Billingshurst, UK) using a microtitre reader (Dynatech MR7000). G6PDH levels were determined as a percentage of the totally disrupted feedstream.

2.6.5 Protein assays

Protein content was determined using a microtitre adaptation of the BCA assay (Pierce- Warriner, Warrington, UK) (Smith et al, 1985). Microtitre plates were incubated at 37°C for 30 min and read at 560nm using a microtitre plate reader (Dynatech MR7000).

In a few cases protein concentrations were also determined by the Bradford assay (Bradford, 1978) using BioRad protein assay reagent. The assay was performed using a microtitre plate according to manufacturer’s instructions, and Bovine Serum Albumin (0- 0.025 mg mL"^) was used as a standard.

2.7 SDS PAGE

SDS PAGE was used to determine the protein content and purity of samples obtained from process purification steps. Samples were concentrated using Trichloroacetic acid (TCA) precipitation.

2.7.1 Trichloroacetic acid (TCA) precipitation

The sample to be concentrated was adjusted to a volume of 1 mL by adding RO water. 0.333 mL of 100% TCA (BDH Chemicals) was added to give a final concentration of 25% (v/v), and the tube was then mixed by inversion and the sample stored at 4°C for 2 h (or overnight). The sample was then spun for 7 min at 14,900 x g for small quantities of visible protein precipitate and 7,500 x g for large amounts of protein precipitate. The supernatant was then decanted, and 1 mL of acetone/5 mM HCl was added. The pellet was then broken up by vortex mixing and the suspension was centrifuged as before. This supernatant was then decanted and 1 mL of acetone was added, the pellet was mixed and centrifuged as before. The supernatant was decanted and the pellets dried using a rotary evaporator (Savant SpeedVac SC 100, Life Sciences International, Basingstoke, UK). The pellets were then stored at -20°C or redissolved in sample buffer.

2.7.2 Gel electrophoresis

All protein gels were run according to the method of Laemmli, (1970). Samples were prepared by adding sample buffer (10-40 pL) (0.5 M Tris/HCl pH 6.8, 10% SDS, 10% glycerol, 5% P-mercaptoethanol (BDH), 0.05% bromophenol blue, and RO water). The buffer was added to the protein in a 1:1 ratio for liquid protein samples or to the required volume if TCA treated (10-40 pL). The gels were run for 1-2 h (Model 400L power pack, Gibco), and, following separation, the gels were treated in fixing solution (acetic acid, 7% v/v; methanol, 40% v/v; RO water, 53% v/v) for 1 h followed by addition of Coomassie blue stain (Coomassie blue, 0.25% w/v; methanol, 45.5% v/v; acetic acid, 9% v/y; RO water, 45.5% v/v) until the band became visible. The gels were then treated with destain (acetic acid, 10% v/v; methanol, 25% v/v; RO water, 65% v/v) until the stain had been removed from the gel leaving clear bands. The gels were photographed and archived using a gel documentation system (Ultraviolet Products Ltd, Cambridge, UK). (Protein standards were; (78, Ovotransferrin (hen egg); 64, Albumin; 42, Ovalbumin (hen egg); 30, Carbonic anhydrase (bovine erythrocyte); 17.2, Myoglobin (equine); 12.3, Cytochrome c (equine).