Enhancement, production, and immobilization of beta-glucosidase from Zobellella denitrificans VIT SB117 and its utilization in bioethanol production from lignocellulosic feedstock

Abstract

β-Glucosidase from novel bacteria Zobellella denitrificans VIT SB117 was isolated, and to increase the production of the enzyme, various growth parameters of the bacteria were optimized. Plackett–Burman design and response surface methodology helped determine the most significant parameters (fructose, temperature, and culture volume) resulting in a 10-fold increase in enzyme activity. The enzyme was purified and kinetics study for free and immobilized enzyme revealed K_m of 4.76 mM and 8.39 mM, K_cat of 255.02 s⁻¹ and 114.02 s⁻¹, and V_max of 4.33 mg/s and 1.25 mg/s, respectively. Enzyme characterization determined optimum substrate concentration and incubation time as 3.5 mM and 10 min respectively for the free enzyme, and 4 mM and 20 min respectively for the immobilized enzyme for maximum activity. pH 5, 45 °C incubation temperature and addition of Mg²⁺ and Mn²⁺ ions exhibited similar stimulatory effects on free and immobilized enzyme activities while Hg²⁺ ions showed strong inhibitory effects. The immobilized enzyme had negligible loss of activity after a month’s storage at 2–4 °C in acetate buffer and ~ 27.76% residual activity after 17 continuous cycles. These optimized parameters were employed for bioethanol production from lignocellulosic wastes. A total cellulose recovery of 52.77% was achieved after pretreatment. Release of ~ 57 mg/g substrate reducing sugars was achieved by enzymatic hydrolysis using immobilized cellulase enzyme complex that produced ~ 5.46 mg/ml bioethanol after 144 h of fermentation using yeast.