A: Blue Native PAGE followed by HNL activity assay. (
a) I: Concentrated fractions of anion exchange purification were applied separately on BN PAGE. NativeMark ™ Unstained Protein Standard (Thermo Fisher Scientific); total protein extract from D. tyermannii leaves (1); flow through (2); elution fractions (3–8); active bands at 20 kDa are highlighted in the box. ( a) II: HNL activity is depicted by the blurred blue spot corresponding to purification fractions 2–7. The total protein extract shows a weak signal (1). Assay conditions: 100 mM citrate buffer pH 4.0; substrate: racemic mandelonitrile. Incubation time 8 min. ( b) Screening of putative HNL sequences. Cell free lysate of E. coli TOP 10 F’ strains expressing six putative HNL proteins. Each sample was tested in triplicate: 30 μL (1), 20 μL (2), 10 μL (3) of cell free lysate, respectively. E. coli TOP 10F’ transformed with pMS vector was used as negative control. Assay conditions: 100 mM citrate buffer pH 4.5; substrate: racemic mandelonitrile 13 mM. The intensive blue spots developed after few seconds of incubation. Proteins with unknown function were named as the relative isotig or contig number found in the transcriptome. ( c) Nucleotide and amino acid sequence of isotig 02643 ( DtHNL1). Fragments detected by mass spectrometry are labeled. Indicated results were obtained from the protein bands excised from lanes 4 (red) and 5 (red and blue). The peptides identified by mass spectrometry cover 72% of the open reading frame.