Proteinase k from tritirachium album

p-type silicon wafers with an <100> orientation (resistivity 0.01–0.02 mΩ cm) were purchased from Siegert Wafer, Aachen, Germany. Hydrofluoric acid (48% aqueous) was obtained from VWR Chemicals, Darmstadt, Germany. PLA (M_w = 18,000–24,000 g mol⁻¹), proteinase K from tritirachium album (≥30 units mg⁻¹), acetone (99.8%), ethyl acetate (99.7%), hexane (99%), isopropanol (99.5%), ethylene glycol (99%) and tris(hydroxymethyl)aminomethane were purchased from Sigma Aldrich, Steinheim, Germany. Sodium hydroxide (98.8%) was purchased from ChemSolute, Renningen, Germany. P. aeruginosa PAO1 (ATCC 15692, isolated from infected wounds) [42 (link),43 (link)]). Luria–Bertani medium (LB broth), LB agar, methanol (99.9%), chloroform (99%) and ethanol (96%) were purchased from Carl Roth, Karlsruhe, Germany. Milli-Q water was obtained from Millipore Direct Q 8 system (MILLIPORE, Schwalbach, Germany) with a resistivity of 18 MΩ cm.

Proteinase K from Tritirachium album was purchased from Sigma (catalogue No. P6556). The lyophilized powder was dissolved to 60 mg ml⁻¹ in 50 mM HEPES pH 7.0. 5 µl protein solution was mixed with 5 µl precipitant solution (1 M NaNO₃, 0.1 M sodium citrate pH 6.5) prior to deposition on the chip.

Five available proteases with therapeutic importance were used: pure elastase from human leukocytes (Sigma-Aldrich, St. Louis, MO, USA E8140), cathepsin B from bovine spleen (cysteine-peptidase) (Sigma-Aldrich, C6286), α-chymotrypsin (serine peptidase) from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA C3142), collagenase from Clostridium histolyticum (metalloproteinases) (Sigma-Aldrich, St. Louis, MO, USA C2674), and thrombin from bovine plasma (serine protease) (Sigma-Aldrich, St. Louis, MO, USA T7513). Six commercially available proteases were also used: esperase from Bacillus sp. (serine-type protease) (Novozyme, Sigma-Aldrich, St. Louis, MO, USA P5860), proteinase K from Tritirachium album (serine protease) (Sigma-Aldrich, St. Louis, MO, USA P2308), subtilisin from Bacillus licheniformis (Subtilisin A is a member of the Serine S8 endoproteinase family) (Sigma-Aldrich, St. Louis, MO, USA P5380), and Aspergillus oryzae (fungal protease/peptidase complex produced by submerged fermentation of a selected strain of Aspergillus oryzae that contains both endoprotease and exopeptidase activities) (Sigma-Aldrich, St. Louis, MO, USA P6110), Bacillus licheniformis (endoprotease of the serine type) (Sigma-Aldrich, St. Louis, MO, USA P4860), and Bacillus sp. (a serine-type protease) (Sigma-Aldrich, St. Louis, MO, USA P3111).

Dispersin B from Aggregatibacter actinomycetemcomitans (20 μg ml⁻¹, Kane Biotech Inc., Manitoba, Canada), Proteinase K from Tritirachium album (100 μg ml⁻¹, Sigma, St Louis, MO, USA) and DNase I (100 U ml⁻¹, Roche Diagnostics, Mannheim, Germany) were used for dispersal of preformed biofilms or degradation of ECM components.

We lysed asynchronous P. falciparum-infected erythrocytes with 0.15% saponin (Sigma-Aldrich, USA) for 5 min and washed them with 1× PBS (diluted from 10× PBS Liquid Concentrate, Gibco, USA). We then lysed parasites with 0.1% Sarkosyl Solution (Bioworld, bioPLUS, USA) in the presence of 1 mg/ml proteinase K (from Tritirachium album, Sigma-Aldrich, USA) overnight at 37 °C. We extracted nucleic acids with phenol/chloroform/isoamyl alcohol (25:24:1) pH 8.0 (Sigma-Aldrich, USA) using 2 ml light Phase lock Gels (5Prime, USA). Lastly, we precipitated the DNA with ethanol using the standard Maniatis method [60 ].

Sorghum cultivars, TX430, were harvested from the University of Queensland farm (Gatton, QLD, Australia). A sorghum flour (SF) sample from whole grains of sorghum was prepared by milling through a 0.5 mm screen using a WonderMill pin mill (WonderMill Company, Pocatello, ID, USA) and was stored in a glass jar at room temperature. The cooked sorghum flour samples were obtained through Rapid visco analyzer (RVA) processing, which has been widely used as a suitable model for simulating thermal processes on a small scale under controlled conditions, as well as monitoring changes in the viscosity as the manufacturing process progresses [37 (link)]. Trypsin from porcine pancreas (Type IX-S, 13,000~20,000 BAEE units/mg protein), papain from Carica papaya (≥3 U/mg), and proteinase K from Tritirachium album (≥30 units/mg protein) used for protein removal were purchased from Sigma Chemical Co. (St. Louis, MO, USA). α-Amylase (PPA, A3176, type VI-B from porcine pancreas, 11 U/mg) and a total starch (AA/AMG) assay kit were purchased from Megazyme International Ireland Ltd. (Bray Co., Wicklow, Ireland). Other chemical reagents were all of analytical grade.