2. Materials and methods
2.1 Sequence collection, alignment and phylogenetic analysis
Sequences were collected by searching the key word “DUF1957” at the National Center for Biotechnology Information (NCBI) on 04th February 2020. To filter redundant and short sequences, the source database and the sequence length were set at RefSeq and 400-1,200 amino acid residues, respectively. Partial and hypothetical sequences were deleted manually. Finally, 2,497 sequences with the title “DUF1957 domain-containing protein” or “Glycoside hydrolase family 57 protein” were used in the sequence alignment and phylogenetic analysis. ClustalW multiple alignment was conducted with the software package MEGA-X26 by setting gap opening penalty at 10, the gap extension penalty at 0.2, the negative matrix at off, and a 30 % delay divergent cutoff. A phylogenetic tree was constructed using the Jones-Taylor-Thornton (JTT) model with the maximum likelihood method in default setting in the software package MEGA-X26, 27. Five conserved sequence regions were analyzed by calculating possibilities of residues within conserved sequence regions based on the multiple sequence alignment, as described previously28.
2.2 Identification of flexible loop and two beta sheets
Protein structures were visualized using the Pymol software29. The three-dimensional structures ofT. thermophilus (PDB ID: 3P0B)6, T. kodakarensis (PDB ID: 3N8T)10, P. horokoshii(PDB ID: 5WU7)19 and T. maritima GBE (AmyC) (PDB ID: 2B5D)20 were downloaded from the Protein Data Bank (PDB). The distances between two residues was measured with the measurement command in Pymol. The flexible loop and the two beta sheet regions were defined based on the crystal structures.
2.3 Overexpression and enzyme production
Various putative GH57 GBEs were overexpressed in Escherichia coli(Table 1), and several mutants were also constructed (Table 2). Genes with optimized codons for overexpression in Escherichia coli were synthesized and then ligated in the pRSET B (T. kodakarensis GBE,T. thermophilus GBE and T. maritima GBE) or pET 28a(+) (the remaining GBEs used in this study) vector by Genscript (USA). Plasmids were transformed into E. coli BL21 (DE3) by subjecting competent cells to a heat shock at 42oC for 90 seconds. A single colony was selected from ampicillin or kanamycin-containing agar plate for further cultivation. E. colistrains carrying the target gene were stored in 25% glycerol stock at -80oC. E. coli BL 21(DE3) carrying the native or mutant gbe was grown in Luria-Bertani (LB) medium (Becton, Dickinson and Company, USA) at 37oC and 150 rpm. When the optical density reached 0.6-0.8 (600 nm), protein expression was induced at 20oC for 12 hours by adding Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Thermo Fisher Scientific, USA) to a final concentration of 0.2 mM. In order to enhance the solubility of the target protein, the chaperone plasmid pG-KJE8 (Takara, Japan) was co-expressed in E. coli by following the manufacture’s protocol. Cells were harvested by centrifugation at 8,000×g for 10 min at 4oC, washed cells twice with 50 mM phosphate buffered saline (PBS) buffer (pH 7.0), and finally resuspended in 5 mM PBS (pH7.0). Cells were disrupted by high pressure homogenizer (Avestin, Canada) at room temperature, with an air pressure of 8 bar and a valve pressure of 6 bar. Heat treatment was performed at 50oC (Calidithermus timidus GBE,Meiothermus silvanus GBE, Petrotoga mexicana GBE) or 65oC (the remaining GBEs used in this study) for 15 minutes to denature E. coli host proteins. Then the supernatant was incubated with nickel metal resin, followed by removal of non-specifically bound host protein by applying a washing buffer containing 25 mM imidazole (Sigma-Aldrich, USA) and elution of the target protein with 250 mM imidazole. Imidazole was removed using a desalting column (Thermo Fisher Scientific, USA) and a storage buffer to 5 mM PBS (pH7.0). Purity of the target protein was checked by SDS-PAGE. Protein concentration was assayed by Bradford (Bio-Rad, USA) using bovine serum albumin as standard.
2.4 Determination of catalytic activity
The branching reaction was performed at 50oC in 5 mM PBS buffer (pH7.0) using 0.125% amylose V (Avebe, The Netherlands) as substrate, and a final concentration of enzyme ranging from 0.03 mg/ml to 0.13 mg/ml was used depending on the total activity of enzyme. Samples were taken at time intervals to monitor the reaction progress. The reaction was stopped by boiling samples at 100oC for 10 min. Branched samples were treated with Pseudomonas sp isoamylase (0.4 U/ml) and Klebsiella planticola pulullanase M1 (1.4 U/ml) (Megazyme, Ireland) at 40oC for 24 h with constant mild shaking. Reducing ends were quantified by the 2,2’-bicinchoninic acid (BCA) method using glucose as standard30. The activity unit was calculated based on reducing end profiles of samples before and after debranching. One unit of activity is defined as 1 μmol reducing ends released or transferred per minute under aforementioned reaction conditions.