Microflex lrf

Fvan-cmp and Gm-cmp activity on corn ChitA was tested as detailed previously (Naumann et al., 2015 ▸ ) by adding protease to solutions containing 1 mM ChitA in buffer (10 mM sodium acetate pH 5.2) followed by incubation at 30°C for 1 h prior to analysis by SDS–PAGE or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The N-terminal peptides released by the polyglycine hydrolase proteolytic activity were assayed by MALDI-TOF MS essentially as described previously (Naumann et al., 2015 ▸ ). The instrument used was a Bruker Daltonics Microflex LRF (Bruker Daltonics, Billerica, Massachusetts, USA) with a pulsed N₂ laser (337 Hz, 60 Hz pulse, 3000 shots) and with reflectron acquisition. The matrix used was 2,5-dihydro­benzoic acid (2,5-DHB). Mass analysis was performed using Peptide Mass Calculator v.3.2 (https://rna.rega.kuleuven.be/masspec/pepcalc.htm).
β-Lactamase activity was tested using the colorimetric substrate nitrocefin as described previously (O’Callaghan et al., 1972 ▸ ). For purified Fvan-cmp, 200 nM enzyme was incubated with substrate for 24 h at 30°C. For mutants, cell-free medium was concentrated tenfold by ultrafiltration and added at 10% of the assay volume. No activity was observed.

Thin layer chromatography (TLC) was used to detect the obtained products of pectin degradation. A 1.5 µL of the culture supernatants, and 1 mM solution of mono-galacturonic acids as a standard solution (purchased from Sigma) were spotted on silica gel 60 F254 (Merck). Chromatography was performed twice in n-butanol/acetic acid/water (2:1:2) as a mobile phase. For visualization, the dried spots on silica gel were sprayed with orcinol/sulfuric acid reagent (8 mg orcinol in 10 mL of 70% sulfuric acid). The plates were heated at 100 °C for 10 min^{32 (link)}.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS):
The oligosaccharides were analyzed by MALDI-TOF MS (Bruker Daltonics Microflex LRF). The samples (1 mL) were lyophilized and dissolved in water (100 μL). The soluble fraction of the samples were mixed with a matrix solution. The matrix solution was prepared by dissolving 50 mg of 2,5-dihydroxybenzoic acid (Sigma Aldrich) in 1 mL of 70% acetonitrile solution. The spectrum for each sample was obtained by collecting 100 shots per spot at five spots. Oligosaccharides were identified based on their theoretical m/z values with the addition of proton, Na⁺, K⁺, and NH₄^{+48 (link)}.