<![CDATA[Abstract. Mardina V, Febriani, Saidi N, Santosa SF, Iqbalsyah TM. 2026. Optimization of thermohalostable α-amylase production from a marine extremophile Geobacillus sp. PLS 41 using response surface methodology and enzymatic characterization. Biodiversitas 27 (9): d270919. https://doi.org/10.13057/biodiv/d270919. α-Amylases are vital in many industries, but most conventional forms denature under the high temperatures and salinity common in industrial processes. This study optimized α-amylase production by the marine thermophilic bacterium Geobacillus sp. PLS 41 using Response Surface Methodology (RSM), followed by enzyme purification and biochemical characterization. Ammonium sulfate precipitation and DEAE Sepharose ion-exchange chromatography were used for enzyme purification. The central composite design evaluated starch concentration, NaCl concentration, temperature, and incubation time. The validated optimum conditions were 2.5% (w/v) starch, 2.5% (w/v) NaCl, 65°C, and 36 h, yielding 38.53±3.00 U/mL, approximately 3.6-fold higher than the initial condition. Ammonium sulfate fractionation at 40-60% saturation produced the highest specific activity of 42.6 U/mg, while DEAE Sepharose chromatography increased the specific activity to 206.6 U/mg, corresponding to 12.07-fold purification and 1.0% yield. The purified enzyme indicated an apparent molecular mass of approximately 63 kDa. Maximum activity occurred at 65°C and pH 7.0. Activity increased to 139.5% in 3 M NaCl, while thermal preincubation retained 104.9% activity after 100 min and reached 122.0% after 80 min. Under 3 M NaCl, residual activity increased to 171.1% after 100 min. Fe²⁺, Mn²⁺, and K⁺ enhanced activity, whereas EDTA and PMSF reduced it. Overall, the enzyme demonstrates strong thermo- and salt-tolerance under laboratory conditions, supporting further evaluation for detergent, starch-processing, kinetic, and scale-up applications.]]>
