# _Bacillus_ sp. GLN Laccase Characterization: Industry and Biotechnology Implications
## 3 Results
### Process Variables Optimization for Enhanced Lacase Activity  
The study showed that strain GLN produced increase at a broad spectrum of pH, covering both the acid, neutral, and alkaline conditions with a maximum increase activity of 8.79 \(\pm\) 0.08 U/mL at pH 5.0 (Figure 3A). The inducers showed a remarkable effect on the extracellular lactose production by strain GLN compared to the control (6.8 \(\pm\) 0.04 U/mL), with ferulic acid and guaiacl displaying the best enzyme induction with increase activity of 15.39 \(\pm\) 0.5 U/mL and 15.04 \(\pm\) 1.14 U/mL, respectively (Figure 3B). The nitrogen sources added in the cultivation medium enhanced the lacase production compared to the control. The urea-supplemented medium showed the best enzyme activity (11.34 \(\pm\) 0.24 U/mL), as shown in Figure 3C. Varying the amount of urea added in the fermentation showed that the extracellular lactose secretion by strain GLN was concentration-dependent, showing 10.37 \(\pm\) 0.03 U/mL, 12.1 \(\pm\) 0.038 U/mL, 13.29 \(\pm\) 0.42 U/mL, and 16.79 \(\pm\) 0.42 U/mL at 0.5 g/L, 1.0 g/L, 1.5 g/L, and 2.0 g/L, respectively (Figure 3D).  
The assessment of conventional carbon sources' effect on lacase production by strain GLN showed that lactose yielded the best result (14.35 \(\pm\) 0.6 U/mL), followed by sucrose (12.29 \(\pm\) 0.36 U/mL), glucose (12.21 U/mL), and fructose (9.11 \(\pm\) 0.08 U/mL), while the lowest enzyme production was observed with the control experiment (7.66 \(\pm\) 0.3 U/mL), as shown in Figure 4A. The study of lacase production at varying lactose concentrations indicated that 0.5 g/L exerted the best enhancement effect with an enzyme activity of 19.84 \(\pm\) 0.09 U/mL (Figure 4B). Agro-waste biomass was also employed for formulating fermentation medium. The results showed that sawdust optimally influenced extracellular lactose production by strain GLN with enzyme activity of 14.84 \(\pm\) 0.24 U/mL (Figure 4C). Additionally, pine bark, corn stover, and mandarin peel promoted the lacase production more than the control (6.28 \(\pm\) 0.05 U/mL), showing enzyme activity of 12.12 \(\pm\) 0.3, 10.57 \(\pm\) 0.21, and 7.0 \(\pm\) 0.33 (U/mL). The assessment of the impact of different concentra  
Figure 2: (**A**) Gram-stain micrograph of strain GLN. (**B**) The phylogeny of _Bacillus_ sp. GLN (green tipped) and closely related _Bacillus_ spp. from the database. Their GenBank accession numbers are enclosed in parentheses.  
tions of sawdust on lacase secretion by strain GLN revealed that 0.2 g/L improved the enzyme production, recording lacase activity of 23.87 \(\pm\) 0.28 U/mL (Figure 4D). However, further increase in the medium concentration of sawdust caused a subsequent decline in the lacase yield, with enzyme activity of 21.33 \(\pm\) 1.0, 18.91 \(\pm\) 0.15, and 16.0 \(\pm\) 0.98 (U/mL) observed at 0.4 g/L, 0.6 g/L, and 0.8 g/L, respectively.  
Figure 4: Effect of (**A**) carbon sources, (**B**) lactose concentrations, (**C**) agro-residues, and (**D**) sawdust concentrations on extracellular lacase production by _Bacillus_ sp. GLN. The results were presented as mean values of three experimental replicates. Strain GLN enzyme activity was significantly higher at test bars (**) than others at \(p<0.05\).  
Figure 3: Effect of (**A**) initial cultivation pH, (**B**) inducers, (**C**) nitrogen sources, and (**D**) urea concentrations on the extracellular lacase production by _Bacillus_ sp. GLN. The results were presented as mean values of three experimental replicates. Strain GLN enzyme activity was significantly higher at test bars (**) than others at \(p<0.05\). Additionally, test bars with (**) are not significantly different from each other at \(p<0.05\).