| Name | : Dina Clarissa Kurniawan |
| Student Number | : 22/501378/PMU/11210 |
| Study Program | : Master’s of Biotechnology |
| : dina.c@mail.ugm.ac.id | |
| : https://www.linkedin.com/in/dina-clarissa-kurniawan-09a/ | |
| Thesis Title | : Expression and Characterization of Thermostable Recombinant Alpha-Amylase from Geobacillus sp. DS3 with Escherichia coli BL21(DE3) Host |
| Description | : Continuous efforts are underway to explore new enzyme sources in order to identify enzymes with unique characteristics suitable for various enzymatic processes such as thermostability, halophilicity, and pH stability. The discovery of a novel Geobacillus sp. DS3 strain, isolated from Sikidang Crater in Dieng, has revealed its ability to produce thermostable ?-amylase. To address challenges associated with high-temperature production, recombinant technology was employed to enhance the expression of the thermostable ?-amylase in the E. coli BL21(DE3) host. This study aimed to express, purify, and characterize the novel thermostable ?-amylase for future application in industries. The enzyme was successfully expressed in E. coli BL21(DE3) at 18? for 20 hours with 0.5 mM IPTG induction. The purification with the Ni-NTA column yielded 69.23% from the initial crude enzyme with increased specific activity to 3.6 fold. The recombinant thermostable ?-amylase had a total molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). This enzyme was active from 30-90? and pH 6-8, optimum at 70? at pH 6. The enzyme remained stable for above 30 minutes at 60?. The Km and Vmax values of the enzyme were 324.03 mg/mL and 36.5 U/mg respectively with dextrin as the substrate. As a metalloenzyme, it exhibited the highest activity in the presence of Ca2+. The functionality of the enzyme was confirmed through HPLC analysis. |