Tryptone

The following growth media were used: 2xTY medium (20 g/L Tryptone (Casitose Type-I, HiMedia, Mumbai, India), 10 g/L Yeast Extract (HiMedia, Mumbai, India), 20 g/L NaCl (Fisher Scientific, Loughborough, UK), pH 7.2) containing 100 μg/mL Ampicillin Ampicillin (Fisher Bioreagents, Pittsburg, PA, USA) and supplemented with 1 mM MgSO₄ (Fisher Scientific, Loughborough, UK) and 1% Glucose (Merck KGaA, Gernsheim, Germany) (2xTY-Amp); LB medium (10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L NaCl, pH 7.2) containing 100 μg/mL Ampicillin and supplemented with 1 mM MgSO₄ and 1% Glucose (LB-Amp); ZYP-5052 medium for autoinduction (1% Tryptone, 0.5% Yeast Extract, 25 mM (NH₄)₂SO₄ (Chem-Lab NV, Zedelgem, Belgium₎, 50 mM KH₂PO₄ (Fisher Bioreagents, UK₎, 50 mM Na₂HPO₄ (Fisher Bioreagents, Loughborough, UK), 0.5% glycerol (Sigma, St. Louis, MO, USA), 0.05% Glucose, 0.2% α-lactose (Carl Roth GmbH, Karlsruhe, Germany), 1 mM MgSO₄), containing 100 μg/mL Ampicillin; TYE agar (10 g/l Tryptone, 5 g/l yeast, 8 g/l NaCl, 15 g/l agar), containing100 μg/mL Ampicillin (TYE-Amp).

Protease secretion was determined after growing the strains on 1/5 TSA (15 g/L Tryptone (Roth, Karlsruhe, Germany), 5 g/L Soytone (Merck, Darmstadt, Germany), 5 g/L NaCl, 15 g/L Agar-Agar (Roth, Karlsruhe, Germany)) amended with 10% (w/v) powdered milk (Roth, Karlsruhe, Germany) at 28 °C in constant darkness or constant light (1800 lux). The mutant strains were inoculated in the center of the plate and colony diameter as well as halo diameter were measured every 24 hours for 2 days at 28 °C. A halo appearing around the inoculated area is indicative for exoprotease activity^{105 (link)}. Calculations are based on the ratio halo/mycelium. QM6aΔtmus53 was used as control for every set. At least three biological replicates were analyzed per strain.

Propionibacterium freudenreichii subsp. freudenreichii DSM 20271^T (DSM 20271) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and was kept as cryopreserved stock culture at −80°C in glycerol.
All chemicals were obtained from Sigma-Aldrich (Steinheim, Germany) unless otherwise specified. Tween 80 was purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). [¹⁵N₂]-ammonium sulphate [(¹⁵NH₄)₂SO₄, 99%] was purchased from Eurisotop (Saarbrücken, Germany). Tryptone, yeast extract and agar were purchased from Roth (Karlsruhe, Germany). HPLC-UV grade solvents were obtained from VWR (Ismaning, Germany). LC-MS grade solvents were from Honeywell (Seelze, Germany).

Agonists GW0742 and GW1516 were purchased from Sigma-Aldrich (Taufkirchen, Germany). Nano-HPLC solvents were LC-MS grade (VWR, Darmstadt, Germany), water was purified with a TKA X-CAD system (Thermo Fisher Scientific, Bremen, Germany). Trypsin (cleaving C-terminally of lysine and arginine), GluC (cleaving C-terminally of glutamate and aspartate), and ProTEV Plus (cleaving C-terminally of ENLYFQ(G/S)) were obtained from Promega (Mannheim, Germany). The cross-linker BS²G-D₀/D4 was obtained from Thermo Fisher Scientific (Rockford, IL). The urea-linker was synthesized and purified in-house.[13 (link)] Bpa was obtained from Bachem (Bubendorf, Switzerland). Tryptone, yeast extract, antibiotics, TCEP, and IPTG were purchased from Roth (Karlsruhe, Germany). Iodacetamide, DTT, D-desthiobiotin, DMTMM, and all other chemicals (Sigma-Aldrich, Taufkirchen, Germany) were obtained at the highest available purity.

The synthesis methodology of AgNPs was prepared according to [20 (link),21 (link),22 (link)]. The tested Geobacillus strains were grown aerobically in 100 mL liquid broth with the following composition (in g/L): tryptone (Roth, Karlsruhe, Germany), 10; meat extract (Merck, Rahway, NJ, USA), 5; NaCl (Merck), 5; CaCl₂ (Merck), 2.3 mM; and ZnSO₄ (Merck), 0.91 µM, in 250 mL Erlenmeyer flasks. The cultures were grown in an orbital shaker (Esco, Singapore, Singapore) at 55 °C with aeration at 180 rpm. After 48 h of incubation, cells were separated by centrifugation at 16,000× g for 10 min. Cell-free secretomes were used as material for AgNP synthesis. The secretomes of the target strains were treated with AgNO₃ (Roth) solution at final concentrations of 2 mM. The whole mixtures were incubated in a shaking incubator for 48 h at 55 °C and 200 rpm. The secretomes without AgNO₃ and bacterial growth medium supplemented with 2 mM AgNO₃ were used as controls. After 48 h of incubation, the mixtures were centrifuged at 3000× g for 10 min to remove media components. Then, mixtures were centrifuged at 16,000× g for 15 min to collect AgNPs. To remove unconverted silver ions, the obtained pellets were washed three times with 70% ethanol and three times with deionized water by centrifugation at 16,000× g for 15 min.