Development of 5-fluorocytosine-resistant Bacillus subtilis mutant with enhanced xylose reductase activity for xylitol production from sugarcane bagasse via solid-state fermentation

This study presents an enhanced method for xylitol production from sugarcane bagasse using a mutant strain of Bacillus subtilis resistant to 5-fluorocytosine. The parent strain was first subjected to mutagenesis using methyl methane sulfonate (MMS) at different concentrations (0.5 to 3 mM) and exposure times of 5 to 30 min, followed by screening for xylitol yield. The most productive mutant (MMS-Fc-C5) yielded 11.90 ± 0.6 mg/g of xylitol. The selected mutant and wild strains were optimized for various parameters, including substrate level (3.12 g), moistening medium volume (5 ml), incubation time (72 h), and inoculum size (20%). Scanning electron microscopy (SEM) revealed morphological characteristics of wild-type and mutant strains. Furthermore, the enzyme activity of xylose reductase (XR) was evaluated under varying conditions and enhanced by specific minerals (CaCO3 15 mM, MgSO4 · 7H2O 10 mM). However, the addition of EDTA and Sodium Dodecyl Sulfate (SDS) decreased XR activity. The final mutant strain exhibited 485 ± 4.85 U/g of XR activity, which is 1.15-fold higher than the wild-type. XR was partially purified (70% ammonium sulfate) and characterized (MX: 33 kDa). GC-MS confirmed the presence of xylitol in the fermentation broth.