Thermostable Amylase from Bacterial Isolate ATF2IV8 of Wae Pellae Hot Spring, Sinjai, Indonesia: Production, Partial Purification and Characterisation

Abstract

Amylase enzymes isolated from thermophilic bacteria have significant industrial applications due to their thermostability and ability to mantain catalytic activity at high temperatures. This study aimed to identify the bacterial isolate ATF2IV8 from Wae Pellae hot spring, produce amylase, and characterise its biochemical properties. Molecular identification of the ATF2IV8 through 16S rRNA gene sequencing showed a close relationship with Calidifontibacillus sp. The crude enzyme was fractionated using ammonium sulfate (0% - 20%, 20% - 40%, 40% - 60%, 60% - 80%, and 80% - 100% saturation), with fraction F4 showing the highest activity. Dialysis of the F4 increased the specific activity approximately sixfold compared to the crude extract. Biochemical characterisation revealed an optimal temperature of 70 °C, an optimal pH of 7, and an optimal substrate concentration of 5%. The enzyme retained 67.14% of its activity after 120 min at 60 °C and demonstrated significant pH stability under neutral conditions. Kinetic parameters of the amylase were determined by nonlinear regression, yielding a Vmax of 3.128 U/mL and a Km of 1.218% (R² = 0.9714). Enzyme activity was enhanced by Ca²⁺ and Mg²⁺ ions, while inhibited by Fe³⁺ and Zn²⁺. SDS–PAGE analysis revealed a prominent protein band of approximately 53 kDa, which may be associated with amylase activity. These results suggest that the thermostable amylase from Calidifontibacillus sp. ATF2IV8 has further potential as a candidate for industrial biotechnological applications, including high-temperature starch processing. HIGHLIGHTS This research reports the characterisation of a thermostable amylase from Calidifontibacillus ATF2IV8, contributing to the growing knowledge of thermostable enzymes derived from Indonesian microbial sources. The enzyme exhibits notable catalytic activity at 70 °C, suggesting its potential suitability for high-temperature industrial applications. Isolated from Wae Pellae Hot Spring, Sinjai, Indonesia, this finding indicates that Indonesian geothermal ecosystems may serve as promising sources of novel enzymes with potential industrial relevance. The enzyme demonstrates preliminary characteristics that may support its applicability in sectors such as food processing, bioethanol production, textile manufacturing, and the detergent industry, particularly for processes requiring thermostable catalysts. The identification of Calidifontibacillus ATF2IV8 provides a basis for further investigation into its potential for commercial development, given the reported roles of related genera in industrial enzyme production. GRAPHICAL ABSTRACT

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