Isolation, expression, and in silico profiling of a thermostable xylanase from Geobacillus stearothermophilus strain...

Xylanase is an industrial enzyme with diverse applications, including nutritional supplements, agro-waste valorization, and paper pulp bleaching. This study aims to investigate the production of recombinant thermostable xylanase for converting plant biomass into fermentable sugars, a key step in various industrial processes.

Geobacillus stearothermophilus strain NASA267, a Gram-positive, thermophilic bacterium, was identified as the top xylanase producer from samples collected in Egypt and Saudi Arabia. The xylanase gene xyl267 was successfully cloned from the NASA267 strain and heterologously expressed in E. coli under the control of a Lambda promoter. Optimal expression conditions were determined, with the highest enzyme activity (40 U/ml) achieved after 4 h of induction at 42 ℃. SDS-PAGE analysis revealed that the molecular weight of the recombinant xylanase was approximately 40 kDa, consistent with the calculated molecular weight (38.6 kDa) based on its amino acid sequence (331 aa). Enzyme sequence and structural analysis revealed that xyl267 shows typical TIM barrel fold where Glu134 and Glu241 constitute the enzyme active site. The xyl267 demonstrated optimal activity at 65 ℃ and maintained full stability up to 60 ℃, while it displayed a half-life of 8 min at 80 ℃. It remained stable at - 20 ℃ for up to 50 days and was most active at pH 8. Although the enzyme was active in presence of various salts, solvents, and cations, the exposure to Cu²⁺, Zn²⁺, Mn²⁺, and methanol reduced the enzyme activity by 47%, 37%, 31%, and 8%, respectively. The enzyme was effective in saccharifying agro-waste, particularly pretreated banana peel, which produced the highest sugar content. These findings highlight xyl267s potential for biomass conversion and industrial applications in high-temperature and alkaline environment.

The xyl267 from a NASA strain was cloned and successfully overexpressed in E. coli, producing a ~ 40 kDa recombinant enzyme. It showed optimal activity at 65 ℃, and was most active at pH 8. While it retained activity in various salts and solvents, it was inhibited by some heavy metals. Xyl267 effectively released fermentable sugars from pretreated banana peel, making it a promising candidate for industrial applications in high-temperature, alkaline environments and agro-waste saccharification.