Abstract
The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50°C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50°C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035mM, 11,735µmol/min/mg protein, 5491 (1/s) and 156,885s-1mM-1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.
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