Purity of Biosynthesized Eumelanin via Solid-State Nuclear Magnetic Resonance

Abstract

Eumelanins are a family of biomolecules with a wide range of functionality in nature and tremendous potential for commercial and industrial applications, provided that they can be produced in large quantities. Our group has developed a method to biosynthesize eumelanin with high yield using the marine bacterium Vibrio natriegens. In this work, we use high-resolution ¹H, ¹³C, and ¹⁵N magic angle spinning (MAS) nuclear magnetic resonance (NMR) to characterize the purity of the extracted solid eumelanin as a function of the quality control methods. A key observation is that biomass eumelanin contains a higher level of biocontaminant compared to supernatant eumelanin. Additional insights into the efficiency of the biosynthetic pathway of eumelanin synthesis were obtained by incorporating ¹³C- and ¹⁵N-enriched precursors into the growth medium and subsequent MAS NMR spectra of the eumelanins. These results highlight the usefulness of solid-state NMR for assessing the quality of the eumelanins produced biosynthetically, which is critical for their further application.

1

(a) ¹H MAS NMR spectra for synthetic eumelanin, NRL and CBC eumelanins, and allomelanin, where all spectra are normalized by intensity; (b) comparison of the three ¹H MAS NMR spectra of eumelanins biosynthesized via V. natriegens where the spectra are normalized by mass [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 2 s, and 64–128 for all the samples except for allomelanin, which were 20 kHz, 8 s, and 16, respectively].

2

¹³C CPMAS NMR spectra for five different eumelanin samples (a) normalized by intensity and (b) normalized by mass [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 2 s, and 4096–8192 for all the samples except for allomelanin, which were 20 kHz, 8 s, and 512, respectively].

3

¹³C CPMAS NMR spectra for NRL-produced eumelanin with varying contact time; other melanin samples show similar behavior with varying contact time (see Figure S1) [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 2 s, and 8192, respectively].

4

¹⁵N CPMAS spectra (a) normalized by intensity and (b) normalized by scan and sample mass for different eumelanin samples [the spinning speed, recycle delay, and number of scans were 10 kHz, 2 s, and 7168–81920 for all the samples except for allomelanin, which were 12.5 kHz, 4 s, and 56,320, respectively.]

5

¹H MAS NMR spectra for two extractions, (a) biomass and (b) supernatant, from five different eumelanin preparations, a control without any isotopic enrichment and samples prepared using ¹³C₁-tyrosine, ¹³C₂-tyrosine, ¹⁵N-tyrosine, and ¹³C-glucose with ¹⁵N-NH₄Cl [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 1 s (supernatant samples), −2 s (biomass samples), and 32–128, respectively].

6

¹³C CPMAS NMR spectra for (a) tyrosine, (c) biomass eumelanins, and (d) supernatant eumelanins. Also shown in (b) is the tyrosine chemical structure. All spectra were acquired with a contact time of 1 ms. For ¹³C CPMAS NMR spectra as a function of contact time, see Figure S3 [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 1 s (supernatant samples), −2 s (biomass samples), and 512–32,000, respectively.].

7

Comparison of ¹³C CPMAS NMR spectra for ¹³C₁-labeled biomass eumelanin as a function of time while undergoing ex situ heat treatment at 150 °C [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 1 s, and 4096, respectively.].

8

Reaction scheme for the enzymatic oxidation of tyrosine to eumelanin.

9

¹⁵N CPMAS NMR spectra for (a) biomass eumelanins and (b) supernatant eumelanins [the spinning speed, recycle delay, and number of scans were 12.5 kHz, 2 s, and 4096–200,000, respectively.]

10

¹H (left column), ¹³C (center column), and ¹⁵N (right column) MAS NMR spectra from Figures , , and and normalized by sample massbiomass (black) and supernatant (red).

11

Eumelanin purification schematic.