Production and characterization of a thermostable endo-β-1,3(4)-glucanase for the combined enzymatic degradation of chitin-glucan to produce prebiotic oligosaccharides from mushroom waste

BACKGROUND: Growing global demand for mushrooms means an increased production of solid mushroom waste (SMW), necessitating novel approaches for waste valorization. Waste stalks and misshapen mushrooms are rich in chitin-glucan polymer – a promising source of prebiotics. Tter16 (EC 3.2.1.6) from Thielavia terrestris was cloned, recombinantly expressed in Pichia pastoris, and biochemically characterized. An enzyme cocktail preparation containing Tter16, two other endo-β-1,3(4)-glucanases (Csph16A and Csph16A-ΔC) and one endo-β-1,6-glucanase (Tvir30) (EC 3.2.1.75), was used to degrade the glucan fraction of fungal chitin-glucan with the aim of valorizing bioactive polymers within commercial mushroom waste. RESULTS: Tter16 was optimally active at pH 5.0, 50 °C, and hyperthermostable at 50 °C with a D-value of 33 days. The Tm of Tter16 was determined to be 67.77 °C and the substrate specificity analysis revealed a preference for mixed-linkage β-glucans. Tter16 was found to possess one of the highest turnover numbers recorded for noncellulolytic fungal glucanases, with a value of 1221 s⁻¹. The enzyme cocktail yielded oligosaccharides including a β-1,6-linked disaccharide and β-1,3-linked oligosaccharides DP 2–5, while minimizing the production of glucose at >90 min of hydrolysis of chitin-glucan. A small-scale chitin-glucan hydrolysis study resulted in a 242.5 mg yield of soluble sugars per g of substrate (24.3% yield) after 90 min of hydrolysis. These oligosaccharides were found to support the growth of the probiotic Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) in vitro. CONCLUSION: The biochemical properties of Tter16 are well-suited for the intended application of mushroom waste valorization. Enzymatic hydrolysis of chitin-glucan released β-glucooligosaccharides displaying prebiotic potential. To the best of our knowledge, this is the first account of the cooperative enzymatic degradation of chitin-glucan with the aim of liberating prebiotic β-glucooligosaccharides from solid mushroom waste and their subsequent in vitro application testing.