Measurement

The following information was taken from a study conducted by Kedia et. al., in 2010 and is subject to changes and optimization with all practical factors considered.

Initial Procedure

The culture broth was removed from the fermenter and centrifuged at 17,000 g for 30 minutes to remove the cells. The supernatant obtained was mixed with three volumes of cold ethanol and kept overnight for precipitation. The precipitate was then collected through centrifugation at 17,000 g for 15 minutes, which resulted in crude γ-PGA.

For removing impurities, the crude γ-PGA was re-dissolved in deionized water, dialyzed against deionized water, and lyophilized to obtain pure γ-PGA. To get the γ-PGA in its free acid form, the supernatant was precipitated at pH 2 followed by dialysis and lyophilization.

NMR Spectroscopy

Characterization of the isolated polymer was performed at the University of Birmingham, School of Chemistry by nuclear magnetic resonance (NMR) spectroscopy. The H-NMR spectra were recorded in D₂O with a Bruker DRX500 NMR spectrometer at 500 MHz.

Fourier-transform infrared spectroscopy was carried out on a Mattson FTIR spectrometer fitted with a durascope and KBr beam splitter. Spectra were acquired over 100 scans within the wavenumber range of 400–4000 cm⁻¹.

Mn was determined by Gel Permeation Chromatography (GPC) as provided by Smithers Rapra Technology Limited, Shawbury, UK, which was calibrated using sodium polyacrylic acid. GPC analysis was carried out at a condition of 0.2 M NaNO₃ and 0.01 M NaH₂PO₄ (pH 7) conditions. Operating conditions were as follows: flow rate of 1 mL/min and at 30°C. Polymer Laboratories 'Cirrus 3.0' software was used for data acquisition and analysis.

Fig. 1: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]

Fig. 1: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]
in Medium E with 2.3 mM MnSO4 [1].

Fig. 1: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]

Fig. 2: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]
in Medium E without MnSO4 [1].

Fig. 1: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]

Fig. 3: H-NMR spectrum of γ-PGA synthesized by B. licheniformis [1]
in Medium C (60 g/l glucose) [1].

References

  1. 1. Kedia, G., Hill, D., Hill, R., & Radecka, I. (2010). Production of poly-γ-glutamic acid by Bacillus subtilis and Bacillus licheniformis with different growth media. Journal of Nanoscience and Nanotechnology, 10(9), 5926-5934.https://doi.org/10.1166/jnn.2010.2614