Browsing by Author "Huang, Liyang"

Plant biomass consists largely of polymeric compounds of which diverse polysaccharides are the main components. Ferulic acid is a ubiquitous phenolic phytochemical in plant cell wall and forms the linkage between plant cell wall polymers. Therefore it is a major aspect in the recalcitrance of cell wall against microbial attack. Ferulic acid esterases (FAEs) are hydrolytic enzymes which participate in plant biomass degradation by removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. By using phylogenetic gene prediction strategy, three putative FAE gene models have been detected from the genome of the ascomycete fungus Aspergillus niger. The codon optimized putative FAE encoding genes have been synthetized for heterologous production in Pichia pastoris GS115. In this work, these three FAEs of A. niger, i.e. FAE796, FAE807 and FAE809, were produced in P. pastoris and their biochemical properties were characterized. The properties included substrate profiling, thermostability, pH optimum and solvent tolerance of the recombinant FAEs. The three A. niger FAEs were successfully produced in P. pastoris resulting as approximately 57 kDa molecular mass proteins. Substrate profiling was performed by using a set of 11 synthetic FAE model substrates and substrates for tannase and lipase activity. FAE796 and FAE809 preferred methoxy substrates, and thus were likely to belong to type A class of FAEs. FAE807 had activity towards a wider range of substrates including methyl sinapate, methyl cinnamate, chlorogenic acid and para-nitrophenyl ferulate, suggesting it to belong to type C class of FAEs. In addition, FAE807 had tannase activity which is a novel property described among the FAEs studied so far. The optimal temperature for FAE796, FAE807 and FAE809 were +37 °C, +55 °C and +55 °C, respectively. FAE809 was the most thermostable enzyme, and retained half of its activity up to +60 °C for 60 min. The studied FAEs were most active at pH 4.0-5.0. FAE809 was relatively stable towards the studied solvents retaining 70%-91% of its activity after solvent treatment.