Recombinant Expression and Characterization of a New Endogenous and Halophilic GH13_5 Subfamily α-Amylase from...

Halophilic α-amylases remain active at high concentrations of salt. Therefore, they can be used in starch-processing industries operating under extreme saline conditions, such as the baking and detergent industries, as well as in effluent remediation processes. In the present work, an endogenous halophilic α-amylase from the CAZy GH13_5 subfamily was recombinantly expressed and characterized from a non-halophilic Paenibacillus pabuli isolate. The predicted structural model revealed the three characteristic domains of α-amylases. In addition, the protein contained the seven conserved sequence regions (CSRs) and the catalytic triad (Asp-Glu-Asp) of α-amylases. The 60.1 kDa purified enzyme had an optimum pH of 7.0 and an optimum temperature of 40 °C. The enzyme's T_m was 53.6 °C, but it remained 80% active after 48 h of incubation at 40 °C, at pH 7.0. The purified enzyme used preferentially starch, maltodextrin, and blocked p-nitrophenyl α-D-maltoheptaoside (BpNPG7) as substrates and produced maltose in starch degradation, as evidenced by thin-layer chromatography. The K_M for starch of the purified enzyme was 1.34 mg/mL, and the V_max was 0.04 mg/min of degraded starch. EDTA, Ca²⁺, and Mg²⁺ acted as activators of the purified enzyme, whereas the detergent SDS and several other ions acted as inhibitors. The purified recombinant enzyme exhibited up to 100% increase in activity in the presence of 1.5-3.0 M NaCl. The high expression level (430 µg/mL of culture medium) and the characteristics of the studied enzyme, such as its ability to liquefy starch under high-salt conditions, make it a promissing tool for industrial starch hydrolysis.