Oxidative Activities of TrAA14A on Cellulosic Substrates

The oxidative activities of TrAA14A on various cellulosic substrates including RAC-85, Avicel, mercerized fiber and α-cellulose, or xyloglucan were determined in the reaction mixture (2.0 mL) containing various substrates (5 mg), 1 μM TrAA14A and 1 mM AscA in sodium acetate buffer (pH 5.0, 50 mM) in an incubator at 45 ℃ and 200 rpm for 24 h. The control reaction containing various substrates (5 mg) with AscA (1 mM), or AscA (1 mM) and Cu²⁺ (1 μM), or inactivated TrAA14A (1 μM, boiled at 99 °C for 15 min) only was also performed in parallel under the same condition. To compare the cellulose-oxidizing activity of TrAA14A with AA9 LPMOs, the oxidative activities on different cellulosic substrates of two previously characterized NcLPMO9C and EpLPMO9A were also determined under the same conditions. After the reaction, all samples were boiled at 99 ℃ for 10 min and centrifuged at 10,000 rpm for 10 min. The nonoxidized and oxidized products in the supernatant were then assayed by HPAEC-PAD [48 (link)]. Briefly, HPAEC analysis was performed on a Dionex ICS-6000 system (Dionex, Sunnyvale, CA, USA) equipped with pulsed amperometric detection (PAD) and a CarboPac PA200 analytical column (3 × 250 mm) with a CarboPac PA200 guard column (3 × 50 mm). Products were separated using 0.1 M NaOH in the mobile phase with the concentration of sodium acetate increasing from 0 to 140 mM (14 min), 140 to 300 mM (8 min), 300 to 400 mM (4 min), and then held constant at 500 mM (3 min) before re-equilibration in 0.1 M NaOH (4 min). The flow rate was set to 0.4 mL/min, the column was maintained at a temperature of 30 °C. The oxidation regioselectivity of TrAA14A was determined by analyzing the products generated from RAC-85 using MALDI-TOF MS as described before [45 (link)]. In all analyses, 2,5-dihydroxybenzoic acid (DHB) in acetonitrile 30% (v/v) was used as the matrix. The synergism of TrAA14A with GHs was performed in a reaction mixture (1 mL) by mixed incubation of TrAA14A (1 μM) with EGI (10 μg), CBHI (20 μg), CBHII (20 μg), or Celluclast®1.5L (0.04 U), and 4 mg/mL RAC-85 or mercerized fiber, with 1 mM AscA in sodium acetate buffer (pH 5.0, 50 mM) at 45 ℃ and 1000 rpm for 1 h in a thermomixer. The control reaction in a reaction mixture (1 mL) containing individual TrAA14A (1 μM), EGI (10 μg), CBHI (20 μg), CBHII (20 μg), or Celluclast®1.5L (0.04 U), and 4 mg/mL RAC-85 or mercerized fiber, with 1 mM AscA was performed under same condition. The reaction was stopped by boiling at 99 ℃ for 10 min and centrifuging at 10,000 rpm for 10 min. The reducing sugar in the supernatant was then assayed by DNS. The degree of synergy (DS) of the coupled enzyme mixtures was calculated by Eq. (1): $$\text{DS}={\text{RS}}_{(\text{GH}+Tr\text{AA14A})}/\left({\text{RS}}_{\text{GH}}+{\text{RS}}_{Tr\text{AA14A}}\right)$$ where RS_{(GH+ TrAA14A)} is the reducing sugar released from the enzyme mixture of GH and TrAA14A, and (RS_GH + RS_TrAA14A) is the sum of the reducing sugar released from the single GH enzyme and TrAA14A.