Lucia Dhiantika Witasari

Thermostable α-amylase from Geobacillus sp. DS3, isolated from the Sikidang Crater, Dieng Plateau, Indonesia, was previously purified and characterized. However, production from thermophilic bacteria requires high-temperature cultivation. This study aimed to clone and express the amy gene encoding α-amylase in Escherichia coli BL21(DE3) for easier enzyme production. The amy gene (1638 bp) was amplified via PCR, TA-cloned, and inserted into the pET-SUMO expression vector, which includes an N-terminal His-tag and SUMO-tag to enhance expression and solubility. The recombinant plasmid (pET-SUMO-amy) was transformed into E. coli BL21(DE3) for protein expression. Homology modelling using MOE software and template PDB ID 1HVX (91.5% identity) revealed a reliable 3D structure. Structural analysis showed altered calcium and sodium ion binding compared to the template, with calcium ions interacting with more residues. Docking studies revealed that maltotetraose binding is stabilized by five key residues: Asp268, His272, Trp300, Asn363, and Asp365. The enzyme displayed optimal activity at 70 °C and retained 60% activity at 90 °C. Kinetic parameters showed a low K_m (6.77 mM) and V_max (0.20 U/mL), indicating high substrate affinity. In conclusion, the recombinant α-amylase exhibited thermostability and substrate affinity suitable for industrial applications such as starch liquefaction and porous starch production at elevated temperatures.

The awareness of eco-friendly food packaging has driven the exploration of biopolymer-based edible films. This study focuses on optimizing and characterizing an edible film derived from glucomannan Porang (Amorphophallus oncophyllus), incorporating sorbitol as a plasticizer. The objective is to address the challenges posed by gas permeability and lack of elasticity in glucomannan films, enhancing their overall functionality. The study employed a central composite design to determine the optimal formulation of varying glucomannan and sorbitol concentrations. The resulting optimized formula exhibited a transparent appearance, controlled thickness (0.005 mm), elevated tensile strength (384.48 MPa), and a degree of flexibility (18.02% strain). Barrier and chemical properties reveal a low moisture content (0.014%), controlled water solubility (4.29%), and excellent resistance to water vapor transmission (WVP of 1.04 x 10-10 g.m-1s-1.Pa-1). FTIR and SEM analysis confirmed the absence of new functional groups and provided insights of film’s microstructure. These characteristics make the film suitable for preserving food products by minimizing moisture transfer. The study contributes to the development of sustainable and functional food packaging alternatives. The optimizing edible film demonstrates promising physical, barrier, and chemical properties, making it a potential candidate as a solution for eco-friendly packaging with enhanced functionality.