Abstract

This study optimized the physico-chemical parameters for immobilized cellulolytic β-glucosidase produced by Trichoderma harzianum Rifai strain Th12 using oil palm frond leaves, an agro-industrial waste substrate, via solid state fermentation. The enzyme was evaluated as an eco-friendly alternative to synthetic chemical fungicides for managing Ganoderma boninense, Fomitopsis meliae, Bipolaris sorokiniana and Phoma herbarum, the causal agents of basal stem rot, brown rot, root rot and leaf spot diseases in oil palm, respectively. Optimization of cultivation conditions was performed using Taguchi orthogonal array design based on five factors: pH, incubation period, temperature, surfactant concentration, and enzyme loading, each at four levels. The maximum activity of free β-glucosidase was 30.50U/g. Under optimized conditions-pH 7.0, 6 days incubation, 30 ℃, 2.0mg/mL enzyme concentration, and 0.05%v/v surfactant the immobilized β-glucosidase exhibited a peak activity of 92.40U/g. The linear model generated by Taguchi analysis accurately predicted the optimal cultivation parameters for T. harzianum Rifai Th12. Antifungal efficacy assays revealed that the immobilized β-glucosidase produced inhibition zones of 92.59%, 82.39%, 81.35%, and 86.21% against G. boninense, F. meliae, B. Sorokiniana, and P. herbarum, respectively, under in vitro conditions. The crude immobilized enzyme demonstrated superior antifungal activity compared to synthetic fungicide tested. These findings demonstrate that immobilized cellulolytic β-glucosidase from T. harzianum Rifai Th12 is a viable, sustainable biocontrol agent. It offers a promising alternative to synthetic fungicides for protecting oil palm against major fungal diseases..

Keywords:Immobilization; Biological agent; β-glucosidase; Taguchi orthogonal array design; Enzyme loading; Optimization; Solid state fermentation; Eco-friendly alternative