Characterization of cold-adapted, halotolerant glycoside hydrolase from Antarctic Halomonas sp. KS41843

Abstract Cold-adapted and halotolerant enzymes are promising candidates for industrial biocatalysis because they remain active under low-temperature and high-salinity conditions. However, thermolability, frequently accompanied by low yield and the difficulty of mimicking the extreme natural environments in vitro, remains the main obstacle to their commercial application. In this study, we biochemically characterized and determined the structure of the glycoside hydrolase family 13 (GH13) protein (R41843G) from Halomonas sp. KS41843, a bacterium isolated from the Antarctic Sea, to assess its potential as a valuable biocatalyst in hypersaline environments. The enzyme exhibited maximal activity at 2.5 M NaCl and retained 89.22 ± 2.39% of its activity at 4.0 M NaCl relative to that in the absence of NaCl, indicating remarkable high salt tolerance. Consistent with this, structural analysis revealed abundant surface-exposed acidic residues, which likely support its halotolerance under high-salt conditions. Thermostability analysis indicated that the enzyme exhibited activity at low temperatures, with substantial activity loss above 30 °C. The enzyme was also stable within a broad pH range (4.0–9.0), with optimal stability at pH 6.0. Substrate specificity tests showed a preference for α-1,4-glucosidic linkages, with the highest relative activity toward maltose. Collectively, the integration of cold adaptation, outstanding halotolerance, and a broad pH stability profile highlights the distinctive properties of R41843G among GH13 enzymes. These biochemical and structural features make R41843G a promising biocatalyst for industrial applications under high-salinity and low-temperature conditions, particularly in the hydrolysis of short-chain maltooligosaccharides. Key points • R41843G is a cold-adapted GH13 enzyme with exceptional salt tolerance. • Surface acidic residues likely support stability under hypersaline conditions. • R41843G preferentially hydrolyzes α-1,4-glucosides, particularly maltose.