ENZYMATIC SYSTEMS IN BIOLOGICAL LIGNOCELLULOSE DEGRADERS AND THEIR ROLE IN LIGNIN MODIFICATION

A key step in developing biofuel is pretreatment, as this step remains the bottleneck in designing an efficient and economical technology for producing biofuels from lignocellulosic biomass. Although various pretreatment methods are available for deconstructing lignocellulosic biomass and obtain cellulosic sugars, they have several disadvantages, including high energy requirements and chemical input. Biological pretreatment of biomass using fungi and bacteria has been long studied to overcome the challenges related to thermochemical pretreatment. The dissertation research focuses on the mechanism elucidation and understanding of these systems to make it possible to develop a pretreatment process that could mimic them. This project aimed to elucidate these biological systems from an enzymatic perspective. The project involved a series of three studies.In the first study, the pretreatment of lignocellulosic biomass, wheat straw, by a white-rot fungus was examined. The extracellular proteome of this fungus was investigated and the pretreated biomass was characterized. This allowed the development of a schematic mechanism to explain the strategy of synergism between the ligninolytic enzymes to pretreat the biomass.In the second study, a more evolved and complex organism, the Formosan termite, as a system was used to examine the lignin deconstruction mechanism. The midgut proteome of this system was examined to explore the enzymes that can modify lignin in the biomass. Based on the enzymes identified, a speculative schematic was proposed to explain the specific modifications of the -O-4 lignin bond in the termite gut.In the third and final study, a putative superoxide dismutase in the termite midgut was confirmed through expression studies in bacteria and yeast hosts. The expressed enzyme was purified to determine the enzyme activity. This enzyme was proposed as a ‘novel ligninase’, based on its role in lignin modification with the help of Reactive Oxygen Species (ROS).The findings from these studies will contribute to the design of in-vitro pretreatment methods inspired from biological degraders of biomass. The results also provide insight for further research on the termite proteome as well as characterization of unknown enzymes

Effects of extracellular proteome on wheat straw pretreatment during solid-state fermentation of Phlebia radiata ATCC 64658

Biological pretreatment of lignocellulosic biomass potentially offers a less energy intensive alternative to chemical pretreatment for the reduction of recalcitrance towards cellulolytic enzymes, but possesses some disadvantages, including a slow rate and loss of polysaccharides. This study characterizes the biodegradation of wheat straw by Phlebia radiata, a white rot fungus capable of secreting three major classes of ligninolytic enzymes: lignin peroxidase, manganese peroxidase, and laccase under natural conditions. We investigated the correlation between synergistic action of fungal enzymes and characteristics of the pretreated biomass. The results showed a sequential expression of enzymes over the course of a three-week pretreatment, with members of the peroxidase family being expressed in week one, followed by laccase expression starting in week two which continued until the course of pretreatment. This highlights the synergy of ligninolytic enzymes in the selective degradation pattern for wheat straw. 1H–13C HSQC NMR spectroscopy results demonstrated reduced amounts of syringyl (S) and hydroxyphenyl (H) lignin after pretreatment. Moreover, the reduction in H lignin was also seen in Pyrolysis – GC/MS and FT-IR results. This strongly suggests that this unique lignin modification pattern is associated with P. radiata extracellular proteome, as expressed during the solid-state fermentation (pretreatment) of wheat straw. [Display omitted] •Ligninolytic enzymes of Phlebia radiata work synergistically in wheat straw degradation.•MnP is secreted early in the process, to generate substrate for laccases.•Laccase allows the lignin modification to continue via relatively higher breakdown of H-units than G and S subunits.•Sequential secretion strategy serves the purpose of an efficient biodegradation