Increasing xylanase activity of Bacillus subtilis by atmospheric pressure plasma jet for biomass hydrolysis

Xylanase producing bacteria, Bacillus subtilis, was bombarded by an atmospheric pressure plasma jet (APPJ) and screened for higher catalytic activity. The bacteria were bombarded with plasma of argon or helium with energy of 120 W for a duration of 1-5 min. A mutant with higher xylanase activity was observed under argon plasma treatment at 1 min on media containing xylan as substrate. Subsequently, the xylanase gene from the mutant was sequenced and named MxynA. Sequence analysis revealed only a single missense mutation on the MxynA gene causing amino acid substitution from threonine to serine at position 162 (T162S) within the xylanase protein of the mutant. Consequently, MxynA was subcloned into expression vector, pETDuet-1 under T7 promoter and expressed in E. coli BL21 (DE3). The optimum temperature and pH of MxynA and its parent expressed in E. coli, named CxynA were 60 °C and pH 5, respectively. Moreover, MxynA showed higher xylanase activity approximately 4 fold higher than that of the control upon a wide range of pH and temperature conditions. From kinetic parameters analysis, the mutant showed higher enzyme turnover (k _cat) than the control. The hydrolysis ability of the MxynA enzyme on lignocellulosic wastes, such as rice straw, corncob and para grass was investigated using the released reducing sugar as an indicator. The MxynA enzyme showed a greater amount of reducing sugar released from all lignocellulosic wastes other than the control, particularly from para grass. This study demonstrated that the T162S mutation possibly improved the catalytic efficiency of MxynA.