Potato starch

Proteins from the extracellular phase of Actinoplanes sp. SE50/110 grown in maltose minimal medium were concentrated by use of Amicon® Ultra 15-mL Centrifugal Filters 3K (Merck Millipore) and washed with 20 mM Tris-HCl (pH 7.5). The concentrate was taken up in 800 μL of 20 mM Tris-HCl (pH 7.5). A Bradford assay was performed (Roti®-Nanoquant, Carl Roth) for total protein quantification. In the starch-binding assay, 80 μg of the total protein raw extract was mixed with 200 μL 10%, 7.5%, 5%, 2.5%, 1%, and 0% (w/v) potato starch (VWR, LOT 171054105) in Tris-HCl buffer (20 mM, pH 7.5) and incubated for 1 h at 28 °C. The supernatant and starch fraction were separated by centrifugation. The starch fraction was washed three times in Tris-HCl buffer (20 mM, pH 7.5) and taken up in the same volume as the supernatant fraction. All 12 fractions were analyzed by SDS-PAGE.

The available chlorine content of a 10–15% v/v stock solution of NaOCl (Sigma Aldrich, Gillingham, UK) was verified with a standard iodine/thiosulfate method [34 ]. The chemical reactants used were: (i) Sodium thiosulphate solution (0.2M) which was prepared from 50 g sodium thiosulphate pentahydrate crystals (99.8% purity, Alfa Aesar, Heysham, UK) dissolved in 1L HPLC water (Chromasolv, Sigma Aldrich, Gillingham, UK), and 25 mg of sodium carbonate (VWR, UK) were added to adjust the pH of the solution to alkaline levels, (ii) potassium iodate (0.05M) (Sigma Aldrich, Gillingham, UK) and potassium iodide (0.24M) (Alfa Aesar, Heysham, UK), (iii) sulphuric acid (3M) (Fisher Scientific, Loughborough, UK) and (iv) potato starch (2% w/v) (VWR, Lutterworth, UK).
Titrations (n = 3) of sodium thiosulphate consumption were carried out after the standardisation of the titrating agent with potassium iodate (0.05M) as a primary standard at 0.22M. The concentration of NaOCl stock solution was 1.474M. Stock NaOCl solution was diluted with HPLC water to obtain 2.5% v/v NaOCl (0.34M). The NaOCl solutions were stored in air-tight dark containers at 5^o C until further use.