In nature, aldehyde dehydrogenase (ALDH) is widely distributed and mainly involved in the oxidation of aldehydes. Thermostability is one of the key features for industrial enzymes. The ability of enzymes to withstand a high operating temperature offers many advantages, including enhancing productivity in industries. This study was conducted to understand the structural and biochemical features of ALDH from thermophilic bacterium, Anoxybacillus geothermalis strain D9. The 3D structure of A. geothermalis ALDH was predicted by YASARA software and composed of 24.3% β-sheet located at the center core region. The gene, which encodes 504 amino acids with a molecular weight of ~56 kDa, was cloned into pET51b(+) and expressed in E.coli Transetta (DE3). The purified A. geothermalis ALDH showed remarkable thermostability with optimum temperature at 60 °C and stable at 70 °C for 1 h. The melting point of the A. geothermalis ALDH is at 65.9 °C. Metal ions such as Fe3+ ions inhibited the enzyme activity, while Li+ and Mg2+ enhanced by 38.83% and 105.83%, respectively. Additionally, this enzyme showed tolerance to most non-polar organic solvents tested (xylene, n-dedocane, n-tetradecane, n-hexadecane) in a concentration of 25% v/v. These findings have generally improved the understanding of thermostable A. geothermalis ALDH so it can be widely used in the industry.
Enzyme stability is regarded as an important criterion for an industrial biocatalyst. Aldehyde dehydrogenase (ALDH) from A. geothermalis strain D9 was previously reported to exhibit good thermostability. However, this enzyme is still not suited to use in harsh environments. In this current work, we aim to see the viability of ALDH in terms of stability when immobilized into Seplite LX120. The purified ALDH was successfully immobilized via physical adsorption at 4 h with 1.25 mg/mL enzyme loading. The immobilized ALDH exhibited improved stability compared to free ALDH as the optimum temperature increased up to 80 °C and was stable with temperatures ranging from 30 to 90 °C. It was also stable in broad pH, ranging from pH 4 to pH 12. Moreover, more than 50% of the immobilized ALDH activity was retained after being stored at 25 °C and 4 °C for 9 and 11 weeks, respectively. The reusability of immobilized ALDH is up to seven cycles. The corroboration of ALDH immobilized on the Seplite LX120 was verified via Fourier-transform infrared spectroscopy, scanning electron microscopy, and a reduction in the surface area. The improved features of immobilized ALDH, especially in enzyme stability, are important for future applications.
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