Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable alpha-amylase production. Recombinant technology was used to express thermostable alpha-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 degrees C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of +/- 70 kDa (+/- 58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30-90 degrees C) and pH range (6-8), with optimal activity at 70 degrees C and pH 6 with great stability at 60 degrees C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.