TY  - JOUR
AU  - Almansoori, Ammar Khazaal Kadhim 
AU  - Alsaedi, Alyaa Abdulhussein 
AU  - Yu, Lo Hui 
AU  - Rahim, Rashidah Abdul 
PY  - 2024
TI  - Enhancement of Geobacillus thermodenitrificans Lipase Activity and Binding Affinity by Acetone Treatment: Kinetic and in silico Investigations
JF  - American Journal of Biochemistry and Biotechnology
VL  - 20
IS  - 4
DO  - 10.3844/ajbbsp.2024.323.332
UR  - https://thescipub.com/abstract/ajbbsp.2024.323.332
AB  - Lipase Geobacillus thermodenitrificans (LipGt) is a thermostable enzyme that has an optimal activity temperature of 65°C. It can tolerate various organic solvents and has an improved activity, especially in the presence of acetone. In this study, in-vitro and in-silico studies were carried out to investigate the effect of acetone in enhancing the kinetic and binding affinity of LipGt. The LipGt was produced in a shake flask and then purified using ultrafiltration, affinity chromatography, and gel-filtration chromatography. Its activity was determined by colorimetric assay through absorbance measurement at 715 nm and comparing it with a standard curve. The Lineweaver Burk plot and molecular docking were utilized to study the kinetic and binding properties between LipGt and olive oil in the presence of acetone, respectively. The findings of our experiment demonstrated that the acetone treatment improved Km, Vmax, and Kcat of LipGt from 0.18 µM, 2.56 µmoL/mg/min, and 69.1 min-1 to 0.30 µM, 4.32 µmoL/mg/min and 116.6 min-1. It indicated that the LipGt had a greater catalytic capacity toward the substrate and a higher reaction rate when it was treated with acetone. In the in-silico study, the binding free energy of LipGt was improved by acetone from -4.4 to -5.8 kcal/moL. The number of hydrophobic interactions was doubled with the acetone treatment, which assisted in maintaining the tertiary structure of LipGt and enhancing its activity after 24 h of incubation. Acetone altered the core active site region of LipGt and increased the binding affinity toward olive oil. This study has discovered a basic understanding of the kinetic and binding affinity of LipGt with acetone, attributing it to become a potential biocatalyst in industrial applications that require operation in harsh conditions such as the production of biodiesel.