Cellytic b plus kit

E. coli strain Shuffle Express was transformed with pQE30-based plasmids expressing recombinant EhPDI enzyme variants (see Table 1). Stable transformants were cultured in LB medium, supplemented with ampicillin, and protein expression was induced with 0.1 mM IPTG. Bacterial cells (from 100 mL) were harvested and lysed under native conditions, using the CelLytic B Plus Kit (Sigma-Aldrich). From the soluble fraction, recombinant proteins were purified by Ni-affinity chromatography (The QIAexpressionist, Qiagen). Eluate fractions were analyzed by SDS-PAGE and those containing more than 95% of pure protein were pooled and concentrated/desalted by ultrafiltration, using a Microsep UF Spin Filter (Pall Co.). Protein concentration was determined by performing the BCA colorimetric assay (Sigma-Aldrich), using BSA as standard.

Xylanase activity was measured with EnzChek^® Ultra Xylanase Assay Kit (Life Technologies) according to manufacturer’s instructions. Briefly, cells were grown for 21 hours, reaching similar densities, harvested and lysed with CelLytic^™ B Plus Kit (Sigma-Aldrich). Cell lysate and supernatant from cultures were diluted and 50 μL of dilutions were mixed with 50 μL of xylanase substrate working solution in flat-bottom 96-well microtiter plate (Greiner Bio-One). They were incubated at room temperature for 40 min and the release of reaction products was measured with the ELx808^™ Microplate Reader (BioTek) with the excitation at 360 nm and emission at 460 nm. Total protein content was measured with Novagen^® BCA Protein Assay Kit (Merck) and xylanase activity was normalized to it.