Mass-spectrometic Analysis of the Amylase from the Mediums LBM, M9M, LBN, M9N, LBE and M9E
Photo 6. Mass-spectrometic analysis of Methionine amylase. The peptides found in the mass-spectrometic analysis are colored blue. Cutting points in the trypsin protease are colored black. Methionines are marked red in the amino acid sequence.
Photo 7. Mass-spectrometic analysis of Norleucine amylase. The peptides found in the mass-spectrometic analysis are colored blue. Cutting points in the trypsin protease are colored black. Methionines are marked red in the amino acid sequence.
Methionine positions that are marked with a red star have been proven to include norleucine.
Photo 8. Mass-spectrometic analysis of Norleucine amylase. The peptides found in the mass-spectrometic analysis are colored blue. Cutting points in the trypsin protease are colored black. Methionines are marked red in the amino acid sequence. Methionine positions that are marked with a red star have been proven to include ethionine. Positions marked with a red question have inclusions that are in question.
Photo 6, photo 7 and photo 8 show the summary of the Mass-spectrometic analysis of the amylases in the LBM, M9M, LBN, M9N, LBE and M9E cultures.
The red stars denote the positions in the protein at which norleucine and ethionine inclusion is evidenced. Positions marked with a red question marks have inclusions that are in question.
In general, it can be said that the level of norleucine and ethionine in the amylase preparation is still quite low in comparison to the methionine levels. (see details attached).
But it worked, and that is reason enough to celebrate!
The inclusion levels should be improved in the course of further experiments at the Max-Planck-Institute for Biochemistry. In order to do that, the first step is to place the amylase in a plasmid-based expression system, which has never been attempted with Bacillus subtilis in connection with non-naturally occurring amino acids.