Abstract
This work reports the amy1 gene cloning from Massilia timonae CTI-57, and its successful expression in Escherichia coli Rosetta™ (DE3) from the pTRCHis2B plasmid. The recombinant AMY1 protein had 47 kDa, and its modeled structure showed a monomer composed of three domains. An N-terminal domain with the characteristic (β/α)8-barrel structure of α-amylases, which contained the catalytic amino acid residues. The second domain was small, and the C-terminal domain was similar to those found in the barley α-amylase. A phylogenetic analysis demonstrated a high sequence identity of the studied protein with bacterial and plant α-amylases from the GH13_6 subfamily. This is the first characterized bacterial α-amylase from this glucoside hydrolase subfamily. Besides starch, the enzyme was also active against maltodextrin, amylopectin, and blocked p-nitrophenyl α-d-maltoheptaoside, but could not use β-cyclodextrin or 4-nitrophenyl α-d-glucopyranoside. The K M for highly pure grade soluble starch from potato and V max values were 0.79 mg/mL and 0.04 mg/min, respectively. The calcium ion showed to be essential for the purified enzyme's activity, while EDTA, molybdenum, cobalt, and mercury were strong inhibitors. The enzyme was almost fully active in SDS presence. The enzyme's optimal pH and temperature were 6.0 and 60 °C, respectively, and its denaturation T m was 79 °C. A TLC analysis revealed that glucose and maltose are products of the enzyme's action on starch. In conclusion, this work described the M. timonae GH13_6 subfamily α-amylase, which showed to be thermostable and anionic detergent-resistant.