A Novel Cobalt-Activated Halotolerant α-Amylase with High Specific Activity from Priestia sp. W243 in Kuwait Sabkha for Biotechnological Applications

Sabkhas, hypersaline ecosystems along Kuwait's coastal zone, are extreme environments that harbor diverse halophilic microorganisms with significant biotechnological potential. Despite this, they remain underexplored, particularly in the context of enzymes that can function under high salinity. The aim of this study is to identify bacterial isolates from Kuwait's sabkhas that produce α-amylase under extreme environmental conditions and to purify and characterize the resulting halotolerant α-amylase. Among the seven α-amylase-producing isolates, <i>Priestia</i> sp. W243, isolated from Mina Abdullah, exhibited the highest enzyme production under optimal growth conditions of pH 9.0, 37 °C, and 7.5% NaCl. A novel halotolerant α-amylase with a remarkably high specific activity (8112.1 U/mg) was purified from this isolate using ultrafiltration, ion-exchange chromatography, and gel-filtration. The purified enzyme, with a molecular weight of 25 kDa, showed optimal activity at 40 °C, pH 8, and 3% NaCl. Notably, the enzyme remained active in the absence of salt and up to 15% NaCl, demonstrating exceptional halotolerance. Metal ion profiling revealed that enzyme activity was significantly enhanced by Co²⁺, whereas Ca²⁺ had a comparatively moderate effect on enzyme activity. When the effects of metal chelators were examined, EDTA, a strong metal chelator, inhibited the enzyme. However, the enzyme remained active when Ca²⁺ was specifically removed using EGTA, suggesting that this α-amylase may be a cobalt-dependent metalloenzyme, which is an unusual characteristic among known α-amylases. Additionally, the enzyme retained its catalytic activity under reducing conditions (e.g., in the presence of DTT and β-mercaptoethanol), indicating structural stability is independent of disulfide bonds. These unique properties distinguish this α-amylase from typical salt- or calcium-dependent counterparts and highlight its potential for industrial applications in high-salt food processing, baking, brewing, and environmental remediation.