Cary 600

ATR-FTIR was performed on samples without preparation with the Cary 600 spectrophotometer (Agilent Technologies, Santan Clara, CA, USA). The range used by the IR spectra was 4000–400 cm⁻¹. Spectra were acquired with 128 scans at 4 cm⁻¹ resolution. All data were processed with Origin software. The signal identification was compared with the published literature.

The pooled plant biomass samples were stored in the dark and transported to the University of Münster. Here, the material was cut into approximately 1 cm long pieces by hand, thoroughly mixed and again stored dry and in the dark until further use.
A subsample of the pooled plant material was finely ground using a mixer mill (Retsch MM 400). Then, 2 mg of the finely ground sample were mixed with 200 mg of potassium bromide (KBr, IR grade, Sigma Aldrich, St. Louis, UAS) in a mortar, obtaining a homogenous powder. The powder was placed in a pelleting press and pressed into 13 mm pellets at a load of 8 t. The pellets were immediately transferred into the FTIR spectrometer (Cary 600, Agilent, Santa Clara, CA, USA), and 32 scans of the sample were recorded and averaged to obtain the final infrared absorption spectrum. The spectra were preprocessed in R [42 ] using the function ir_bc() from the R package “ir” [43 ] (version 0.0.0.9000) which is based on a “rubberband” algorithm from the spc.hyperspec() function of the R package hyperSpec [44 ]. To interpret the FTIR spectra, we assigned absorption features to major structural moieties in organic matter as explained in Table 1.

The prepared BP nanosheets and HA-DAH/BP complexes were analyzed by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments Co., Malvern, UK), UV/vis spectrophotometry (S-3100, Scinco Co., Seoul, Korea), Fourier transform - infrared spectroscopy (FT-IR, Cary 600, Agilent Technologies), and transmission electron microscopy (TEM, JEM-1011, JEOL Co., Akishima, Japan). The physical structure of BP nanosheets and HA-DAH/BP complexes was analyzed by TEM, and the surface modification of BP nanosheets with HA was assessed by DLS and FT-IR.

Purified Rv2231c (0.30 mg/ml) was dissolved in Tris-buffer (100 mM NaCl and 20 mM Tris–HCl pH 8.0). Circular dichroism (CD) spectroscopic analysis was performed using a Jasco 1500 CD spectropolarimeter. A quartz cuvette of path length 1 mm was used for the CD measurements. Continuous scanning (20 nm/min) from 240 to 200 nm was performed. Data were collected and processed using the K2D2 software after each sample was scanned three times [26 (link)]. A concentrated Rv2231c protein sample (4 µl) was spotted onto a diamond ATR reflective element. An Agilent Cary 600 instrument with a DTGS detector was used to acquire FTIR spectra with a resolution of 4 cm⁻¹. The absorption mode was set at 128 scans to collect Rv2231c spectra at room temperature compared to the 128 scan single beam spectra of the buffer alone. The second derivative of the spectrum was calculated using MATLAB (MathWorks Inc., Massachusetts, USA) for kinetic applications. The spectrum shows no evidence of water vapor above 1750 cm⁻¹. The band amplitudes of the spectra were normalized and the baseline was fitted using a least-squares curve-fitting program. The secondary structure of Rv2231c was deduced by fitting the curve with amide I region. The second-derivative spectrum was used to locate the peak positions using the Voigt function [27 (link)].