Qiaexpress kit

12 h after gene expression induction the cells were harvested by centrifugation and resuspended in 50 mM Na-Phosphate buffer (pH 6.0) containing 1,5 M ammonium sulfate for the following purification step. The cells were disrupted by french pressure cell, and centrifuged at 20,000×g for 30 min at 4°C. The resulting supernatant was applied to a preequilibrated column of phenyl sepharose (HiLoad 26/10 Phenyl Sepharose HP; GE Healthcare, Munich) for hydrophobic interaction chromatography (HIC) and eluted from the column with a stepwise decreasing gradient of ammonium sulfate. The recombinant protein-containing fractions were pooled, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), concentrated and further purified by size exclusion chromatography (HiLoad 16/60 Superdex 200 prep grade; GE Healthcare, Munich). As a final purification step the pooled fractions resulting from size exclusion chromatography were applied to immobilized metal ion affinity chromatography using Ni²⁺-NTA (“Qiaexpress Kit, Qiagen”). The enzyme was eluted from the column with 50 mM phosphate buffer (pH 6.0) containing 250 mM imidazole. Protein concentration was determined by the method of Bradford using bovine serum albumin (BSA) as a standard [28] . The purified protein was dialyzed against 50 mM phosphate buffer (pH 6.0) and the purity was analyzed by SDS-PAGE.

His₆-tagged proteins from B. subtilis were overexpressed using the QIAexpress kit (Qiagen), inducing with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 5 to 7 h. MaeB from E. coli was directly overexpressed in the growth medium by adding 0.2 mM IPTG. Cells were harvested by centrifugation at 4°C, washed with 0.9% NaCl, resuspended in lysis buffer (100 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM dithiothreitol [DTT], and 4 mM phenylmethylsulfonyl fluoride), and disrupted by three passages through a French press cell at 4°C. Cell-free lysates were obtained by centrifugation for 10 min at 23,000 × g and 4°C. His₆-tagged proteins were purified using Ni²⁺-charged nitrilotriacetic acid (Ni-NTA) affinity columns (GE Healthcare) according to the manufacturer’s instructions. Proteins were eluted from the column with elution buffer (500 mM imidazole), which was subsequently replaced by storage buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM DTT, and 0.5 mM EDTA) (8 (link)) using ultrafiltration columns with a 10-kDa size cutoff (Millipore). The correct size was verified by SDS-PAGE. Proteins were stored at 4°C for, at most, 1 day before performing activity assays.

Wild-type (WT) TrCel12A (Egl3) with signal peptide excluded and the 6 ×His-tag designated at the C-terminal was cloned and inserted into pPIC9k (Invitrogen, Carlsbad, CA, USA), while mutants were constructed by site-directed mutagenesis according to the PCR-based method54 (link). Competent yeast (Pichia pastoris) GS115 cells (Invitrogen) were then transformed with the recombinant plasmids after they were confirmed by DNA sequencing (Biosune, Shanghai, China). The exogenous proteins were induced and purified with the protocols described in the Original Pichia Expression Kit (Invitrogen) and the QIAexpress Kit (Qiagen, Hilden, Germany). All proteins expressed were buffer exchanged in 20 mM acetate buffer (pH 5.5) and the concentration was determined by the Bradford method55 (link). Manipulations to another member of the GH12 family, AnEglA, from Aspergillus niger CBS120.49 (PDB 1KS4) were the same to TrCel12A and the WT AnEglA and its mutants were buffer exchanged in the same acetate buffer with pH 3.8. All chemicals, reagents and enzymes used were of analytical grade from Sigma (St. Louis, MO, USA) or Sangon (Shanghai, China).