A study of selected glucan-degrading enzymes from fungi, and their potential application in the valorisation of mushroom-production waste streams to yield products for animal and human health

Monaghan Mushrooms produces 30,000 tonnes of mushroom waste annually. This study focuses upon the identification, recombinant production, and characterization of glucanase enzymes capable of releasing oligosaccharides from mushroom waste and liberating pure chitin for further processing. Two GH16 endo-β-1,3(4)-glucanases and one GH30 endo-β-1,6-glucanase were expressed in Pichia pastoris. One endo-β-1,3(4)-glucanase (Tter16 from Thielavia terrestris) comprises the Lam16 catalytic domain, while the other GH16 enzyme (Csph16A from Cladosporium sphaerospermum) contains the Lam16A catalytic domain and a C-terminus of unknown function. The β-1,6-glucanase (Tvir30 from Trichoderma virens) is composed of the characteristic GH30 TIM-barrel domain with an associated β-side structure.The enzymes were purified by IMAC or ion exchange chromatography. Notably, Csph16A was purified as two isoforms resulting from differential glycosylation. All three enzymes share a pH optimum of 5.0 and temperature optima ranging from 45 to 55oC, which suits the current chitin-glucan processing conditions at Monaghan Mushrooms (incubation at pH 5.0, 50oC for 24 h). However, only Tter16 displayed appreciable thermostability, with a D-value of 33 days at 50oC. Tvir30 and Csph16A were inactivated within 1 h at 50oC, though Tvir30 demonstrated improved thermal stability at 40 and 45oC. To elucidate the function of the unknown domain and to potentially improve the enzymes’ thermostability, Csph16A was truncated of its C-terminus, which led to an approximately 70% increase in residual activity after 1 h at 50oC. Csph16A was found to be halotolerant up to 1.25 M NaCl, with truncation improving salt tolerance up to 1.5 M NaCl.Tter16 displayed the highest specific activity in this study at 383.9 U/mg on barley β-glucan, with the truncated mutant Csph16A-ΔC coming second at 256.9 U/mg (an improvement compared to both wild-type isoforms). Tvir30 demonstrated a specific activity of 169.9 U/mg on pustulan, a β-1,6-linked glucan polysaccharide.Preliminary applications testing revealed that the synergistic activity of all four enzymes was capable of liberating glucooligosaccharides from fungal chitin-glucan polymer DP ≥ 2 after 90 min of hydrolysis at 50oC, pH 5.0. These oligosaccharides were freeze-dried and were found to support the growth of one out of two lactic acid bacteria tested, indicating their prebiotic potential. The purity of the chitin liberated after 120 min of hydrolysis by the enzyme mix was comparable to that of the commercial enzyme. The enzymes developed in this study therefore have the potential to replace the commercial enzyme to release lucrative glucooligosaccharides and liberate pure chitin from mushroom waste.<br><br>