Horseradish peroxidase delignification of fermented rooibos modifies biomass structural and chemical properties and improves holocellulolytic enzyme cocktail efficacy

The purpose of the study was to pretreat fermented rooibos biomass with partially purified horseradish peroxidase (HRP) for lignin removal and to convert delignified biomass to soluble sugars through saccharification with a formulated holocellulolytic enzyme cocktail (HEC). HRP enzyme was extracted from the horseradish root tissue and was partially purified by membrane filters and characterised biochemically. HRP enzyme was used to pretreat the fermented rooibos biomass to remove lignin before hydrolysing it with the HEC. Our findings indicated that HRP is versatile because it displayed activity on guaiacol, 8-aminoquinoline, and decolourised methylene blue dye. HRP had a pH optimum of 4.5 and displayed a mesophilic temperature range. The kinetics studies indicated that HRP displayed a higher affinity towards guaiacol ( K m = 0.082 mg/mL) followed by 8-aminoquinoline ( K m = 0.221 mg/mL). However, the catalytic efficiency revealed that the enzyme hydrolysed guaiacol (63436.48 s − 1 . mg/mL) and 8-aminoquinoline (59189.81 s − 1 . mg/mL) efficiently. HRP pretreatment of rooibos biomass significantly removed lignin content and increased pores on the surface as visualised with SEM. FTIR validated the SEM results by showing reductions at 3324.81, 1615.16 and 1018.75 cm − 1 , corresponding to crystalline cellulose, lignin and holocellulose regions, respectively. HRP pretreated biomass had the lowest crystallinity index of 11.2% compared to 20% of the control. HRP delignified rooibos biomass was hydrolysed effectively by the HEC, which released about 10% yield of soluble sugars compared to 6% of control. We conclude that HRP pretreatment significantly modified the structural and chemical properties of the biomass, making it more accessible to hydrolytic enzymes.