Cloning and Characterization of Cohnella sp. A01 Carbohydrate-Active Enzymes

DNA extraction kits were purchased from Bioneer Company (Seoul, Korea). The restriction enzymes, NdeI, NotI, and plasmid pTZ57R/T were prepared from Fermentas (Glen Burnie, MD, USA). Plasmid extraction kit was procured from Roche Company (Switzerland). Bacterial strains including E. coli DH5α and E. coli BL21 (DE3), the expression vector pET-26b (+), and Ni-NTA resin were purchased from Invitrogen (Carlsbad, USA) and pustulan from Invivogen Company (San Diego, CA, USA). Laminarin, pullulan, starch, cellulose, and sucrose were obtained from Sigma (St. Louis, USA). Other chemicals utilized in this study were prepared from Merck (Darmstadt, Germany). Cohnella sp. A01 was obtained from shrimp pond waste water at Choebdeh (Abadan, Iran; accession No. JN208862.1)[21 (link)]. VMD 1.9 (University of Illinois, Urbana-Champaign, USA), Gene Runner 4.0 (Lynnon Biosoft, Canada), and Graphpad Prism 6 (San Diego, CA, USA) software were used to analyze protein structure and DNA sequence as well as to draw Michaelis-Menten curve and to calculate K_m and V_max. The phylogenetic tree was established using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT)[22 (link)].