Solid-state NMR spectroscopic studies of 13C,15N,29Si-enriched biosilica from the marine diatom Cyclotella cryptica

Abstract

Diatoms are algae producing micro- and nano-structured cell walls mainly containing amorphous silica. The shape and patterning of these cell walls is species-specific. Herein, the biosilica of Cyclotella cryptica, a centric marine diatom with a massive organic matrix, is studied. Solid-state NMR spectroscopy is applied to gain deeper insight into the interactions at the organic-inorganic interface of the cell walls. The various organic compounds like polysaccharides as well as proteins and long-chain polyamines (LCPAs) are detected by observation of heteronuclei like 13C and 15N whereas the silica phase is studied using 29Si NMR spectroscopy. The sensitivity of the NMR experiments is strongly enhanced by isotope-labeling of the diatoms during cultivation with 13C, 15N and 29Si. The presence of two different chitin species in the biosilica is demonstrated. This observation is supported by a monosaccharide analysis of the silica-associated organic matrix where a high amount of glucosamine is found. Moreover, the Rotational Echo Double Resonance (REDOR) experiment provides distance information for heteronuclear spins. 13C{29Si} REDOR experiments reveal proximities between different organic compounds and the silica phase. The closest contacts between silica and organic compounds appear for different signals in the 13C-chemical shift range of 40–60 ppm, the typical range for LCPAs.

DNP-Supported Solid-State NMR Studies of 13C,15N,29Si-Enriched Biosilica of Cyclotella Cryptica and Thalassiosira Pseudonana

“…2). We observed similar 13 C-13 C correlations as in the standard PDSD experiment 26 , however with considerably higher signal intensities. This enabled the detection of new signals which are not observable in the conventional experiment.…”

“…1, right). As we have discussed elsewhere 26 , C. cryptica contains a massive organic matrix and produces extracellular chitin brils of ca. 50 nm thickness [33][34] (see also Figure S1).…”

“…Indeed, DNP signal enhancements for polyssacharides and polyamines amounted to about 40% to the protein signal enhancements for S. turris 3 . In contrast, the organic layer for C. cryptica is signi cantly larger 26 which results in almost uniform DNP enhancements in 1D 13 C CP MAS data and is possibly the reason for the larger (relative) decrease in DNP enhancement from 26 to 5-8 for carbohydrates.…”

“…A monosaccharide analysis which provides information about the total carbohydrate composition revealed glucose and mannose as main monosaccharides. Xylose, ribose, fucose, galactose and glucosamine are also present in signi cant concentrations 26 . However, a differentiation between monosaccharides and monosaccharide units from polysaccharides is not possible due to the performed hydrolysis step.…”

“…In the present work, we have used DNP-ssNMR to examine isotope-labeled diatoms of Cyclotella cryptica biosilica 26 . In particular, the use of DNP allowed us to study the presence of silica-attached chitin.…”