Lignin

Lignin content at the different developmental stages of prickles was estimated as described previously (Iiyama & Wallis, 1990). Briefly, 300‐mg tissue was homogenized in 50 mM phosphate buffer (pH 7.0) and centrifuged at 1,400 g for 5 min at RT, followed by three washes in 1% Triton × 100 (v/v) in 1 M NaCl (pH 7.0). The resulting pellet was again washed with 1 M NaCl, distilled water, and absolute acetone. The washed pellet was dried at 60℃ for 24 hr, lyophilized, and was used as the PFCW fraction. The PFCW content was measured on the basis of fresh weight of the tissue (mg/g of FW). Twenty milligram of PFCW fraction was incubated with 0.5 ml acetyl bromide (25% v/v in acetic acid), at 70℃ for 30 min in a screw‐cap tube. Tubes were immediately cooled on ice and mixed with solubilization buffer (0.9 ml of 2 M NaOH and 0.1 ml of 0.5 M hydroxylamine HCL). Tubes were centrifuged at 1,400g for 5 min, supernatant was collected, and the absorbance was recorded at 280 nm using a Biospectrometer (Eppendorf, Germany). Lignin content was quantified using a standard curve prepared using 1 mg/ml Lignin (Sigma‐Aldrich, USA; Figure S1a).

Peroxidase was produced through submerged fermentation as previously described by Falade et al. (2017b) , where 100 mL of lignin fermentation medium (LFM): K₂HPO₄ (4.55 g L⁻¹), KH₂PO₄ (0.53 g L⁻¹), MgSO₄ (0.5 g L⁻¹), NH₄NO₃ (5 g L⁻¹), yeast extract (0.1 g L⁻¹) and 0.1 % w/v lignin (Sigma-Aldrich, SA) was inoculated with 2 % bacterial suspension (A_{600 nm} ≈ 0.1) at pH 7 using uninoculated media as control. The culture was subsequently, incubated for 48 h using the conditions for isolation (Falade et al., 2017b ), afterwards, the crude enzyme was prepared as previously reported (Falade et al., 2019a ).