Bark depolymerization during submerged fermentation using monofloral honey, a natural mediator substitute, and integration between laccases vs. bark biopolymers, characterized by Py-GC-MS

Abstract

Due to increasing waste production and disposal problems arising from synthetic polymer production, there is a critical need to substitute materials with biodegradable and renewable resources. Attempts to use laccases as a catalyst to enhance the catalytic properties of enzymes have shown them to be a promising solution for bark depolymerization. In this study, eight different fungal strains were tested for laccase enzyme production during submerged fermentation (SF), and the Pleurotus species were shown to be the best producers among the competing fungal strains. P. pulmonarius mainly produces laccase enzyme in production medium (PM) at initial conditions of pH 5.5 and 30 °C. Bark depolymerization was conducted in SF and we identified polyphenols/polyaromatic compounds after four weeks when the PM was induced with 50 mg per 100 mL of each bark during the lag-phase. During SF where honey was used as a natural mediator substitute (NMS) in the PM, laccase activities were about 1.5 times higher than those found in comparable cultures without honey in the PM. These samples were analyzed by GC-MS/MS. The laccase enzyme was purified using UNO® sphere Q-1 anion exchange chromatography and the molecular weight was determined to be ∼50 kDa on 10% SDS-PAGE. The laccase kinetic parameters V_max, K_m, and turnover number (K_cat) were found to be 76.9 μM min⁻¹, 909 μM and 739 min⁻¹, respectively, from a Lineweaver–Burk plot. Furthermore, laccases are suitable for biotechnological applications that transform bark biomass into high value bark biopolymers/biochemicals. The differences observed among the identified aromatic compound MS/MS profiles were due to the utilization of two different bark species. Py-GC-MS analysis of bark showed differing effects of fungal activity on bark composition. Polyphenolics were separated in reverse-phase mode using HPLC with a pinnacle DB Biphenyl, C18 column, and UV detector. Two recognition wavelengths of 290 and 340 nm were selected to improve the separation of each single compound in monofloral honey and bark-fermented samples. This study is novel because it replaces natural mediators (NM) with monofloral honey in PM and bark materials impregnated with honey, and studies the effects of fungi-derived laccases on bark biopolymers.