Combining LC-MS/MS, PMF and N-terminal amino acid sequencing for multiplexed characterization of a bacterial surfactant glycoprotein biosynthesized by Acinetobacter radioresistens S13?

RSC Advances Pub Date: 2014-02-05 DOI: 10.1039/C4RA00692E

Abstract

Surfactants of biological origin are in demand both in the food industry and for cosmetic applications. In the present paper the glycosylated nature of a surfactant protein, previously identified in the Acinetobacter radioresistens S13 proteome, was demonstrated. A multiplexed analysis was performed to establish its amino acid sequence homology degree with AlnA, a previously characterized surfactant from A. radioresistens KA53. The amino acid sequence coverage study (N-terminal amino acid sequencing, peptide mass fingerprinting and LC-MS/MS experiments) revealed a 99.97% identity, that is the substitution of one amino acid only. Gly25 of A. radioresistens KA53 AlnA is replaced by Thr in the protein identified in A. radioresistens S13. Such change gives rise to Asn-Asp-Thr N-glycosylation consensus sequon, which is absent in A. radioresistens KA53 AlnA. Actually, Asn23 of the A. radioresistens S13 protein could be identified after N-glycosyl hydrolase treatment only. Asn-Asp-Thr is a peculiar glycosylation consensus sequence since Asp in the central position was shown to decrease the protein glycosylation efficiency in eukaryotes. Our findings provide additional support that glycosylation mechanisms in bacteria differ from those observed in eukaryotic cells. The emulsifying activity of the extracellular protein extracts of A. radioresistens S13 (containing the AlnA-like protein) and a commercial solubilizer widely used in cosmetic preparations, i.e. ethoxylated hydrogenated castor oil (EHCO), were compared. A. radioresistens S13 extracellular protein extracts showed greater emulsifying activity on every tested molecule, especially on peppermint essential oil, with respect to EHCO.

Graphical abstract: Combining LC-MS/MS, PMF and N-terminal amino acid sequencing for multiplexed characterization of a bacterial surfactant glycoprotein biosynthesized by Acinetobacter radioresistens S13
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